Fused heterocyclic compounds and their use as sirtuin modulators

ABSTRACT

Provided herein are novel sirtuin-modulating compounds and methods of use thereof. The sirtuin-modulating compounds may be used for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, polyglutamine diseases, ocular diseases and/or disorders, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing. Also provided are compositions comprising a sirtuin-modulating compound in combination with another therapeutic agent.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application PCT/US2006/007744, filed Mar. 3, 2006, whichclaims the benefit of priority to U.S. Provisional Application No.60/658,430, filed Mar. 3, 2005, and 60/705,386, filed Aug. 4, 2005,which applications are hereby incorporated by reference in theirentireties. International Application PCT/US2006/007744 was publishedunder PCT Article 21(2) in English.

BACKGROUND

The Silent Information Regulator (SIR) family of genes represents ahighly conserved group of genes present in the genomes of organismsranging from archaebacteria to a variety of eukaryotes (Frye, 2000). Theencoded SIR proteins are involved in diverse processes from regulationof gene silencing to DNA repair. The proteins encoded by members of theSIR gene family show high sequence conservation in a 250 amino acid coredomain. A well-characterized gene in this family is S. cerevisiae SIR2,which is involved in silencing HM loci that contain informationspecifying yeast mating type, telomere position effects and cell aging(Guarente, 1999; Kaeberlein et al., 1999; Shore, 2000). The yeast Sir2protein belongs to a family of histone deacetylases (reviewed inGuarente, 2000; Shore, 2000). The Sir2 homolog, CobB, in Salmonellatyphimurium, functions as an NAD (nicotinamide adeninedinucleotide)-dependent ADP-ribosyl transferase (Tsang andEscalante-Semerena, 1998).

The Sir2 protein is a class III deacetylase which uses NAD as acosubstrate (Imai et al., 2000; Moazed, 2001; Smith et al., 2000; Tanneret al., 2000; Tanny and Moazed, 2001). Unlike other deacetylases, manyof which are involved in gene silencing, Sir2 is insensitive to class Iand II histone deacetylase inhibitors like trichostatin A (TSA) (Imai etal., 2000; Landry et al., 2000a; Smith et al., 2000).

Deacetylation of acetyl-lysine by Sir2 is tightly coupled to NADhydrolysis, producing nicotinamide and a novel acetyl-ADP ribosecompound (Tanner et al., 2000; Landry et al., 2000b; Tanny and Moazed,2001). The NAD-dependent deacetylase activity of Sir2 is essential forits functions which can connect its biological role with cellularmetabolism in yeast (Guarente, 2000; Imai et al., 2000; Lin et al.,2000; Smith et al., 2000). Mammalian Sir2 homologs have NAD-dependenthistone deacetylase activity (Imai et al., 2000; Smith et al., 2000).Most information about Sir2 mediated functions comes from the studies inyeast (Gartenberg, 2000; Gottschling, 2000).

Biochemical studies have shown that Sir2 can readily deacetylate theamino-terminal tails of histones H3 and H4, resulting in the formationof 1-O-acetyl-ADP-ribose and nicotinamide. Strains with additionalcopies of SIR2 display increased rDNA silencing and a 30% longer lifespan. It has recently been shown that additional copies of the C.elegans SIR2 homolog, sir-2.1, and the D. melanogaster dSir2 genegreatly extend life span in those organisms. This implies that theSIR2-dependent regulatory pathway for aging arose early in evolution andhas been well conserved. Today, Sir2 genes are believed to have evolvedto enhance an organism's health and stress resistance to increase itschance of surviving adversity.

SIRT3 is a homolog of SIRT1 that is conserved in prokaryotes andeukaryotes (P. Onyango et al., Proc. Natl. Acad. Sci. USA 99:13653-13658 (2002)). The SIRT3 protein is targeted to the mitochondrialcristae by a unique domain located at the N-terminus. SIRT3 hasNAD+-dependent protein deacetylase activity and is ubiquitouslyexpressed, particularly in metabolically active tissues. Upon transferto the mitochondria, SIRT3 is believed to be cleaved into a smaller,active form by a mitochondrial matrix processing peptidase (MPP) (B.Schwer et al. J. Cell Biol. 158: 647-657 (2002)).

Caloric restriction has been known for over 70 years to improve thehealth and extend the lifespan of mammals (Masoro, 2000). Yeast lifespan, like that of metazoans, is also extended by interventions thatresemble caloric restriction, such as low glucose. The discovery thatboth yeast and flies lacking the SIR2 gene do not live longer whencalorically restricted provides evidence that SIR2 genes mediate thebeneficial health effects of this diet (Anderson et al., 2003; Helfandand Rogina, 2004). Moreover, mutations that reduce the activity of theyeast glucose-responsive cAMP (adenosine 3′5′-monophosphate)-dependent(PKA) pathway extend life span in wild type cells but not in mutant sir2strains, demonstrating that SIR2 is likely to be a key downstreamcomponent of the caloric restriction pathway (Lin et al., 2001).

SUMMARY

Provided herein are novel sirtuin-modulating compounds and methods ofuse thereof.

In one aspect, the invention provides novel sirtuin-modulating compoundsof Formula (I):

or a salt thereof, where, as valence permits:

Ring A is optionally substituted;

L is absent, substituted or unsubstituted phenylene, substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene,substituted or unsubstituted pyrazolylene, substituted or unsubstitutedbenzothiazolylene, —NR₄—, —C(O)O—, —C(O)NR₄—, —NR₄C(O)—, —NR₄—C(O)—NR₅—,—S—, —CHR₆═CHR₇— or —CHR₆—C(O)—;

L′ is absent, substituted or unsubstituted phenylene, substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene,substituted or unsubstituted pyrazolylene, substituted or unsubstitutedbenzothiazolylene, substituted or unsubstituted indenedionylene,—C(O)O—, —C(O)NR₄—, —NR₄C(O)—, —NR₄—C(O)—NR₅—, —S—, —CHR₆═CHR₇— or—CHR₈—C(O)—, provided that at least one of L and L′ is substituted orunsubstituted phenylene, substituted or unsubstituted —O-phenylene,substituted or unsubstituted thienylene, substituted or unsubstitutedpyrazolylene, substituted or unsubstituted benzothiazolylene,substituted or unsubstituted indenedionylene, —NR₄—, —C(O)O—, —C(O)NR₄—,—NR₄C(O)—, —NR₄—C(O)—NR₅—, —S—, —CHR₆═CHR₇— or —CHR₈—C(O)—;

R₁ is absent, —H, —NR₄R₅, —N₄C(O)R₅, —OR₅, naphthyl or a heterocyclicgroup, provided that L and R₁ are not both absent unless X is N;

R₂ is —H, unsubstituted alkyl, —NR₄R₅, —NR₄C(O)R₅, —OR₅, substituted orunsubstituted phenyl, naphthyl or a heterocyclic group;

R₃ is —H, —NR₄R₅, —N₄C(O)R₅, —OR₅ or a substituted or unsubstitutedheterocyclic group, or R₂ and R₃, taken together with the atoms to whichthey are attached, form an optionally substituted heterocyclic group, orR₃ is absent when Z is O or S;

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group;

R₆, R₇ and R₈ are independently selected from the group consisting ofhalogen, —OR₄, —CN, —CO₂R₄, —OCOR₄, —OCO₂R₄, —C(O)NR₄R₅, —OC(O)NR₄R₅,—C(O)R₄, —COR₄, —SR₄, —OSO₃H, —S(O)_(n)R₄, —S(O)_(n)OR₄, —S(O)_(n)NR₄R₅,—NR₄R₅, —NR₄C(O)OR₅, —NR₄C(O)R₅ and —NO₂;

W is C or N;

X is C or N;

Y is C or N;

Z is C, N, O or S, provided that at least two of W, X, Y and Z are C;and

n is 1 or 2.

In a second aspect, the invention provides novel sirtuin-modulatingcompounds represented by Structural Formula (Ia):

wherein:

R₁₀ is selected from —H, —C(O)—N(R₄₀)(R₅₀), —S(O)₂N(R₄₀)(R₅₀), or—CH₂—N(R₄₀)(R₅₀); wherein each of R₄₀ and R₅₀ is independently selectedfrom —H, —C₁-C₃ straight or branched alkyl, —(C₁-C₃ straight or branchedalkyl)-N(CH₃)₂, —(C₁-C₃ straight or branched alkyl)-heterocyclyl,—(C₁-C₃ straight or branched alkyl)-alkylheterocyclyl, or wherein R₄₀and R₅₀ taken together with the N atom to which they are bound form a5-6 membered heterocyclic ring that is optionally substituted with—(C₁-C₃ straight or branched alkyl), and wherein at least one of R₄₀ orR₅₀ is not H;

R₁₁, is selected from —C₁-C₃ straight or branched alkylene or —C(O)—;and

each of ring K and ring E is independently substituted with up to threesubstituents independently selected from halo, —CF₃, —O—(C₁-C₃ straightor branched alkyl), —S—(C₁-C₃ straight or branched alkyl), —N(R₄₀)(R₅₀),—S(O)₂—N(R₄₀)(R₅₀), heterocyclyl, (C₁-C₃ straight or branchedalkyl)-heterocyclyl, —O—(C₁-C₃ straight or branched alkyl)-heterocyclyl,—S—(C₁-C₃ straight or branched alkyl)-heterocyclyl, or is optionallyfused to a 5-6 membered heterocyclyl or heteroaryl, wherein anyheterocyclyl or heteroaryl is optionally substituted with —C₁-C₃straight or branched alkyl.

In a further aspect, the invention provides novel sirtuin-modulatingcompounds of Formula (Ib):

wherein:

Z is selected from O or S;

R₁₀ is selected from —H, C(O)—N(R₄₀)(R₅₀), —S(O)₂N(R₄₀)(R₅₀), or—CH₂—N(R₄₀)(R₅₀); wherein each of R₄₀ and R₅₀ is independently selectedfrom —H, —C₁-C₃ straight or branched alkyl, —(C₁-C₃ straight or branchedalkyl)-N(CH₃)₂, —(C₁-C₃ straight or branched alkyl)-heterocyclyl,—(C₁-C₃ straight or branched alkyl)-alkylheterocyclyl, or wherein R₄₀and R₅₀ taken together with the N atom to which they are bound form a5-6 membered heterocyclic ring that is optionally substituted with—(C₁-C₃ straight or branched alkyl), and wherein at least one of R₄₀ orR₅₀ is not H.

R₁₁ is selected from —C₁-C₃ straight or branched alkylene or —C(O)—;

each of R₁₂ and R₁₃ is independently selected from —H or —(C₁-C₃straight or branched alkyl), or R₁₂ and R₁₃ are taken together to form abenzene ring that is substituted with up to two substituentsindependently selected from —(C₁-C₃ straight or branched alkyl), —CF₃ orhalo; and

ring K is substituted with up to three substituents independentlyselected from halo, —CF₃, —O—(C₁-C₃ straight or branched alkyl),—S—(C₁-C₃ straight or branched alkyl), —N(R₄₀)(R₅₀), —S(O)₂—N(R₄₀)(R₅₀),heterocyclyl, (C₁-C₃ straight or branched alkyl)-heterocyclyl, —O—(C₁-C₃straight or branched alkyl)-heterocyclyl, —S—(C₁-C₃ straight or branchedalkyl)-heterocyclyl, or is optionally fused to a 5-6 memberedheterocyclyl or heteroaryl, wherein any heterocyclyl or heteroaryl isoptionally substituted with —C₁-C₃ straight or branched alkyl.

In another aspect, the invention provides novel sirtuin-modulatingcompounds of Formula (II):

or a salt thereof, where:

Rings C, D and E are optionally substituted; and

x is 0 or 1.

In yet another aspect, the invention provides novel sirtuin-modulatingcompounds of Formula (VII):

or a salt thereof, where:

Ring F is optionally substituted;

L′ is substituted or unsubstituted phenylene, substituted orunsubstituted thienylene, —C(O)O—, —S—, —CHR₆═CHR₇— or —CHR₈—C(O)—;

R₂ is —H, unsubstituted alkyl, —NR₄R₅, —NR₄C(O)R₅, —OR₅ or aheterocyclic group;

R₃ is —H, —NR₄R₅, —N₄C(O)R₅, —OR₅ or a heterocyclic group, or R₂ and R₃,taken together with the atoms to which they are attached, form anoptionally substituted heterocyclic group, or R₃ is absent when Z is Oor S;

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group;

R₆, R₇ and R₈ are independently selected from the group consisting ofhalogen, —OR₄, —CN, —CO₂R₄, —OCOR₄, —OCO₂R₄, —C(O)NR₄R₅, —OC(O)NR₄R₅,—C(O)R₄, —COR₄, —SR₄, —OSO₃H, —S(O)_(n)R₄, —S(O)_(n)OR₄, —S(O)_(n)NR₄R₅,—NR₄R₅, —NR₄C(O)OR₅, —NR₄C(O)R₅ and —NO₂;

Z is C, N, O or S; and

n is 1 or 2.

The invention also includes salts, prodrugs and metabolites of thecompounds disclosed herein.

Also provided are pharmaceutical compositions comprising one or morecompounds of Formulas (I)-(X) or a salt, prodrug, or metabolite thereof.

In another aspect, the invention provides methods for usingsirtuin-modulating compounds, or compostions comprisingsirtuin-modulating compounds. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be used for a variety of therapeutic applications including, forexample, increasing the lifespan of a cell, and treating and/orpreventing a wide variety of diseases and disorders including, forexample, diseases or disorders related to aging or stress, diabetes,obesity, neurodegenerative diseases, chemotherapeutic inducedneuropathy, neuropathy associated with an ischemic event, oculardiseases and/or disorders, cardiovascular disease, blood clottingdisorders, inflammation, and/or flushing, etc. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay also be used for treating a disease or disorder in a subject thatwould benefit from increased mitochondrial activity, for enhancingmuscle performance, for increasing muscle ATP levels, or for treating orpreventing muscle tissue damage associated with hypoxia or ischemia. Inother embodiments, sirtuin-modulating compounds that decrease the leveland/or activity of a sirtuin protein may be used for a variety oftherapeutic applications including, for example, increasing cellularsensitivity to stress, increasing apoptosis, treatment of cancer,stimulation of appetite, and/or stimulation of weight gain, etc. Asdescribed further below, the methods comprise administering to a subjectin need thereof a pharmaceutically effective amount of asirtuin-modulating compound.

In certain aspects, the sirtuin-modulating compounds may be administeredalone or in combination with other compounds, including othersirtuin-modulating compounds, or other therapeutic agents.

DETAILED DESCRIPTION

1. Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule (such as a nucleicacid, an antibody, a protein or portion thereof, e.g., a peptide), or anextract made from biological materials such as bacteria, plants, fungi,or animal (particularly mammalian) cells or tissues. The activity ofsuch agents may render it suitable as a “therapeutic agent” which is abiologically, physiologically, or pharmacologically active substance (orsubstances) that acts locally or systemically in a subject.

The term “bioavailable” when referring to a compound is art-recognizedand refers to a form of a compound that allows for it, or a portion ofthe amount of compound administered, to be absorbed by, incorporated to,or otherwise physiologically available to a subject or patient to whomit is administered.

“Biologically active portion of a sirtuin” refers to a portion of asirtuin protein having a biological activity, such as the ability todeacetylate. Biologically active portions of a sirtuin may comprise thecore domain of a sirtuin. Biologically active portions of SIRT1 havingGenBank Accession No. NP_(—)036370 that encompass the NAD+ bindingdomain and the substrate binding domain, for example, may includewithout limitation, amino acids 62-293 of GenBank Accession No.NP_(—)036370, which are encoded by nucleotides 237 to 932 of GenBankAccession No. NM_(—)012238. Therefore, this region is sometimes referredto as the core domain.

Other biologically active portions of SIRT1, also sometimes referred toas core domains, include about amino acids 261 to 447 of GenBankAccession No. NP_(—)036370, which are encoded by nucleotides 834 to 1394of GenBank Accession No. NM_(—)012238; about amino acids 242 to 493 ofGenBank Accession No. NP_(—)036370, which are encoded by nucleotides 777to 1532 of GenBank Accession No. NM_(—)012238; or about amino acids 254to 495 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 813 to 1538 of GenBank Accession No. NM_(—)012238.

The term “companion animals” refers to cats and dogs. As used herein,the term “dog(s)” denotes any member of the species Canis familiaris, ofwhich there are a large number of different breeds. The term “cat(s)”refers to a feline animal including domestic cats and other members ofthe family Felidae, genus Felis.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “conserved residue” refers to an amino acid that is a member ofa group of amino acids having certain common properties. The term“conservative amino acid substitution” refers to the substitution(conceptually or otherwise) of an amino acid from one such group with adifferent amino acid from the same group. A functional way to definecommon properties between individual amino acids is to analyze thenormalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz, G. E. and R. H. Schirmer.,Principles of Protein Structure, Springer-Verlag). According to suchanalyses, groups of amino acids may be defined where amino acids withina group exchange preferentially with each other, and therefore resembleeach other most in their impact on the overall protein structure(Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure,Springer-Verlag). One example of a set of amino acid groups defined inthis manner include: (i) a charged group, consisting of Glu and Asp,Lys, Arg and His, (ii) a positively-charged group, consisting of Lys,Arg and His, (iii) a negatively-charged group, consisting of Glu andAsp, (iv) an aromatic group, consisting of Phe, Tyr and Trp, (v) anitrogen ring group, consisting of His and Trp, (vi) a large aliphaticnonpolar group, consisting of Val, Leu and Ile, (vii) a slightly-polargroup, consisting of Met and Cys, (viii) a small-residue group,consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro, (ix) analiphatic group consisting of Val, Leu, Ile, Met and Cys, and (x) asmall hydroxyl group consisting of Ser and Thr.

“Diabetes” refers to high blood sugar or ketoacidosis, as well aschronic, general metabolic abnormalities arising from a prolonged highblood sugar status or a decrease in glucose tolerance. “Diabetes”encompasses both the type I and type II (Non Insulin Dependent DiabetesMellitus or NIDDM) forms of the disease. The risk factors for diabetesinclude the following factors: waistline of more than 40 inches for menor 35 inches for women, blood pressure of 130/85 mmHg or higher,triglycerides above 150 mg/dl, fasting blood glucose greater than 100mg/dl or high-density lipoprotein of less than 40 mg/dl in men or 50mg/dl in women.

A “direct activator” of a sirtuin is a molecule that activates a sirtuinby binding to it. A “direct inhibitor” of a sirtuin is a moleculeinhibits a sirtuin by binding to it.

The term “ED₅₀” is art-recognized. In certain embodiments, ED₅₀ meansthe dose of a drug which produces 50% of its maximum response or effect,or alternatively, the dose which produces a pre-determined response in50% of test subjects or preparations. The term “LD₅₀” is art-recognized.In certain embodiments, LD₅₀ means the dose of a drug which is lethal in50% of test subjects. The term “therapeutic index” is an art-recognizedterm which refers to the therapeutic index of a drug, defined asLD₅₀/ED₅₀.

The term “hyperinsulinemia” refers to a state in an individual in whichthe level of insulin in the blood is higher than normal.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “insulin resistance” refers to a state in which a normal amountof insulin produces a subnormal biologic response relative to thebiological response in a subject that does not have insulin resistance.

An “insulin resistance disorder,” as discussed herein, refers to anydisease or condition that is caused by or contributed to by insulinresistance. Examples include: diabetes, obesity, metabolic syndrome,insulin-resistance syndromes, syndrome X, insulin resistance, high bloodpressure, hypertension, high blood cholesterol, dyslipidemia,hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke,coronary artery disease or myocardial infarction, hyperglycemia,hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance,delayed insulin release, diabetic complications, including coronaryheart disease, angina pectoris, congestive heart failure, stroke,cognitive functions in dementia, retinopathy, peripheral neuropathy,nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome,hypertensive nephrosclerosis some types of cancer (such as endometrial,breast prostate, and colon), complications of pregnancy, poor femalereproductive health (such as menstrual irregularities, infertility,irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy,cholesterol related disorders, such as gallstones, cholescystitis andcholelithiasis, gout, obstructive sleep apnea and respiratory problems,osteoarthritis, and prevention and treatment of bone loss, e.g.osteoporosis.

The term “livestock animals” refers to domesticated quadrupeds, whichincludes those being raised for meat and various byproducts, e.g., abovine animal including cattle and other members of the genus Bos, aporcine animal including domestic swine and other members of the genusSus, an ovine animal including sheep and other members of the genusOvis, domestic goats and other members of the genus Capra; domesticatedquadrupeds being raised for specialized tasks such as use as a beast ofburden, e.g., an equine animal including domestic horses and othermembers of the family Equidae, genus Equus.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, livestock animals (including bovines, porcines, etc.),companion animals (e.g., canines, felines, etc.) and rodents (e.g., miceand rats).

The term “naturally occurring form” when referring to a compound means acompound that is in a form, e.g., a composition, in which it can befound naturally. For example, since resveratrol can be found in redwine, it is present in red wine in a form that is naturally occurring. Acompound is not in a form that is naturally occurring if, e.g., thecompound has been purified and separated from at least some of the othermolecules that are found with the compound in nature. A “naturallyoccurring compound” refers to a compound that can be found in nature,i.e., a compound that has not been designed by man. A naturallyoccurring compound may have been made by man or by nature.

A “naturally occurring compound” refers to a compound that can be foundin nature, i.e., a compound that has not been designed by man. Anaturally occurring compound may have been made by man or by nature. Forexample, resveratrol is a naturally-occurring compound. A “non-naturallyoccurring compound” is a compound that is not known to exist in natureor that does not occur in nature.

“Obese” individuals or individuals suffering from obesity are generallyindividuals having a body mass index (BMI) of at least 25 or greater.Obesity may or may not be associated with insulin resistance.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion.

A “patient”, “subject”, “individual” or “host” refers to either a humanor a nonhuman animal.

The term “percent identical” refers to sequence identity between twoamino acid sequences or between two nucleotide sequences. Identity caneach be determined by comparing a position in each sequence which may bealigned for purposes of comparison. When an equivalent position in thecompared sequences is occupied by the same base or amino acid, then themolecules are identical at that position; when the equivalent siteoccupied by the same or a similar amino acid residue (e.g., similar insteric and/or electronic nature), then the molecules can be referred toas homologous (similar) at that position. Expression as a percentage ofhomology, similarity, or identity refers to a function of the number ofidentical or similar amino acids at positions shared by the comparedsequences. Expression as a percentage of homology, similarity, oridentity refers to a function of the number of identical or similaramino acids at positions shared by the compared sequences. Variousalignment algorithms and/or programs may be used, including FASTA,BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCGsequence analysis package (University of Wisconsin, Madison, Wis.), andcan be used with, e.g., default settings. ENTREZ is available throughthe National Center for Biotechnology Information, National Library ofMedicine, National Institutes of Health, Bethesda, Md. In oneembodiment, the percent identity of two sequences can be determined bythe GCG program with a gap weight of 1, e.g., each amino acid gap isweighted as if it were a single amino acid or nucleotide mismatchbetween the two sequences.

Other techniques for alignment are described in Methods in Enzymology,vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996),ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co.,San Diego, Calif., USA. Preferably, an alignment program that permitsgaps in the sequence is utilized to align the sequences. TheSmith-Waterman is one type of algorithm that permits gaps in sequencealignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAPprogram using the Needleman and Wunsch alignment method can be utilizedto align sequences. An alternative search strategy uses MPSRCH software,which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithmto score sequences on a massively parallel computer. This approachimproves ability to pick up distantly related matches, and is especiallytolerant of small gaps and nucleotide sequence errors. Nucleicacid-encoded amino acid sequences can be used to search both protein andDNA databases.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified, such as by conjugation with a labeling component. Theterm “recombinant” polynucleotide means a polynucleotide of genomic,cDNA, semisynthetic, or synthetic origin which either does not occur innature or is linked to another polynucleotide in a nonnaturalarrangement.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration of a drug to a host. If it is administeredprior to clinical manifestation of the unwanted condition (e.g., diseaseor other unwanted state of the host animal) then the treatment isprophylactic, i.e., it protects the host against developing the unwantedcondition, whereas if administered after manifestation of the unwantedcondition, the treatment is therapeutic (i.e., it is intended todiminish, ameliorate or maintain the existing unwanted condition or sideeffects therefrom).

The term “protecting group” is art-recognized and refers to temporarysubstituents that protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed by Greene and Wuts inProtective Groups in Organic Synthesis (2^(nd) ed., Wiley: New York,1991).

The term “pyrogen-free”, with reference to a composition, refers to acomposition that does not contain a pyrogen in an amount that would leadto an adverse effect (e.g., irritation, fever, inflammation, diarrhea,respiratory distress, endotoxic shock, etc.) in a subject to which thecomposition has been administered. For example, the term is meant toencompass compositions that are free of, or substantially free of, anendotoxin such as, for example, a lipopolysaccharide (LPS).

“Replicative lifespan” of a cell refers to the number of daughter cellsproduced by an individual “mother cell.” “Chronological aging” or“chronological lifespan,” on the other hand, refers to the length oftime a population of non-dividing cells remains viable when deprived ofnutrients. “Increasing the lifespan of a cell” or “extending thelifespan of a cell,” as applied to cells or organisms, refers toincreasing the number of daughter cells produced by one cell; increasingthe ability of cells or organisms to cope with stresses and combatdamage, e.g., to DNA, proteins; and/or increasing the ability of cellsor organisms to survive and exist in a living state for longer under aparticular condition, e.g., stress (for example, heatshock, osmoticstress, high energy radiation, chemically-induced stress, DNA damage,inadequate salt level, inadequate nitrogen level, or inadequate nutrientlevel). Lifespan can be increased by at least about 20%, 30%, 40%, 50%,60% or between 20% and 70%, 30% and 60%, 40% and 60% or more usingmethods described herein.

“Sirtuin-activating compound” refers to a compound that increases thelevel of a sirtuin protein and/or increases at least one activity of asirtuin protein. In an exemplary embodiment, a sirtuin-activatingcompound may increase at least one biological activity of a sirtuinprotein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplarybiological activities of sirtuin proteins include deacetylation, e.g.,of histones and p53; extending lifespan; increasing genomic stability;silencing transcription; and controlling the segregation of oxidizedproteins between mother and daughter cells.

“Sirtuin-inhibiting compound” refers to a compound that decreases thelevel of a sirtuin protein and/or decreases at least one activity of asirtuin protein. In an exemplary embodiment, a sirtuin-inhibitingcompound may decrease at least one biological activity of a sirtuinprotein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplarybiological activities of sirtuin proteins include deacetylation, e.g.,of histones and p53; extending lifespan; increasing genomic stability;silencing transcription; and controlling the segregation of oxidizedproteins between mother and daughter cells.

“Sirtuin-modulating compound” refers to a compound of Formulas (I)-(X)as described herein. In exemplary embodiments, a sirtuin-modulatingcompound may either up regulate (e.g., activate or stimulate), downregulate (e.g., inhibit or suppress) or otherwise change a functionalproperty or biological activity of a sirtuin protein. Sirtuin-modulatingcompounds may act to modulate a sirtuin protein either directly orindirectly. In certain embodiments, a sirtuin-modulating compound may bea sirtuin-activating compound or a sirtuin-inhibiting compound.

“Sirtuin protein” refers to a member of the sirtuin deacetylase proteinfamily, or preferably to the sir2 family, which include yeast Sir2(GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank AccessionNo. NP_(—)501912), and human SIRT1 (GenBank Accession No. NM_(—)012238and NP_(—)036370 (or AF083106)) and SIRT2 (GenBank Accession No.NM_(—)012237, NM_(—)030593, NP_(—)036369, NP_(—)085096, and AF083107)proteins. Other family members include the four additional yeastSir2-like genes termed “HST genes” homologues of Sir two) HST1, HST2,HST3 and HST4, and the five other human homologues hSIRT3, hSIRT4,hSIRT5, hSIRT6 and hSIRT7 (Brachmann et al. (1995) Genes Dev. 9:2888 andFrye et al. (1999) BBRC 260:273). Preferred sirtuins are those thatshare more similarities with SIRT1, i.e., hSIRT1, and/or Sir2 than withSIRT2, such as those members having at least part of the N-terminalsequence present in SIRT1 and absent in SIRT2 such as SIRT3 has.

“SIRT1 protein” refers to a member of the sir2 family of sirtuindeacetylases. In one embodiment, a SIRT1 protein includes yeast Sir2(GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank AccessionNo. NP_(—)501912), human SIRT1 (GenBank Accession No. NM_(—)012238 orNP_(—)036370 (or AF083106)), and human SIRT2 (GenBank Accession No.NM_(—)012237, NM_(—)030593, NP_(—)036369, NP_(—)085096, or AF083107)proteins, and equivalents and fragments thereof. In another embodiment,a SIRT1 protein includes a polypeptide comprising a sequence consistingof, or consisting essentially of, the amino acid sequence set forth inGenBank Accession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096,NP_(—)036369, or P53685. SIRT1 proteins include polypeptides comprisingall or a portion of the amino acid sequence set forth in GenBankAccession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369,or P53685; the amino acid sequence set forth in GenBank Accession Nos.NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369, or P53685 with 1to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative aminoacid substitutions; an amino acid sequence that is at least 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos.NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369, or P53685, andfunctional fragments thereof. Polypeptides of the invention also includehomologs (e.g., orthologs and paralogs), variants, or fragments, ofGenBank Accession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096,NP_(—)036369, or P53685.

“SIRT3 protein” refers to a member of the sirtuin deacetylase proteinfamily and/or to a homolog of a SIRT1 protein. In one embodiment, aSIRT3 protein includes human SIRT3 (GenBank Accession No. AAH01042,NP_(—)036371, or NP_(—)001017524) and mouse SIRT3 (GenBank Accession No.NP_(—)071878) proteins, and equivalents and fragments thereof. Inanother embodiment, a SIRT3 protein includes a polypeptide comprising asequence consisting of, or consisting essentially of, the amino acidsequence set forth in GenBank Accession Nos. AAH01042, NP_(—)036371,NP_(—)001017524, or NP_(—)071878. SIRT3 proteins include polypeptidescomprising all or a portion of the amino acid sequence set forth inGenBank Accession AAH01042, NP_(—)036371, NP_(—)001017524, orNP_(—)071878; the amino acid sequence set forth in GenBank AccessionNos. AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878 with 1 toabout 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acidsubstitutions; an amino acid sequence that is at least 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos.AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878, and functionalfragments thereof. Polypeptides of the invention also include homologs(e.g., orthologs and paralogs), variants, or fragments, of GenBankAccession Nos. AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878.In one embodiment, a SIRT3 protein includes a fragment of SIRT3 proteinthat is produced by cleavage with a mitochondrial matrix processingpeptidase (MPP) and/or a mitochondrial intermediate peptidase (MIP).

The term “substantially homologous” when used in connection with aminoacid sequences, refers to sequences which are substantially identical toor similar in sequence with each other, giving rise to a homology ofconformation and thus to retention, to a useful degree, of one or morebiological (including immunological) activities. The term is notintended to imply a common evolution of the sequences.

The term “synthetic” is art-recognized and refers to production by invitro chemical or enzymatic synthesis.

The terms “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” areart-recognized and refer to the administration of a subject composition,therapeutic or other material other than directly into the centralnervous system, such that it enters the patient's system and, thus, issubject to metabolism and other like processes.

The term “therapeutic agent” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. The term also means any substance intended for use in thediagnosis, cure, mitigation, treatment or prevention of disease or inthe enhancement of desirable physical or mental development and/orconditions in an animal or human.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. The therapeuticallyeffective amount of such substance will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art. For example, certain compositions described herein may beadministered in a sufficient amount to produce a desired effect at areasonable benefit/risk ratio applicable to such treatment.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operable linked. Inpreferred embodiments, transcription of one of the recombinant genes isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of genes as described herein.

“Treating” a condition or disease refers to curing as well asameliorating at least one symptom of the condition or disease.

A “vector” is a self-replicating nucleic acid molecule that transfers aninserted nucleic acid molecule into and/or between host cells. The termincludes vectors that function primarily for insertion of a nucleic acidmolecule into a cell, replication of vectors that function primarily forthe replication of nucleic acid, and expression vectors that functionfor transcription and/or translation of the DNA or RNA. Also includedare vectors that provide more than one of the above functions. As usedherein, “expression vectors” are defined as polynucleotides which, whenintroduced into an appropriate host cell, can be transcribed andtranslated into a polypeptide(s). An “expression system” usuallyconnotes a suitable host cell comprised of an expression vector that canfunction to yield a desired expression product.

The term “vision impairment” refers to diminished vision, which is oftenonly partially reversible or irreversible upon treatment (e.g.,surgery). Particularly severe vision impairment is termed “blindness” Or“vision loss”, which refers to a complete loss of vision, vision worsethan 20/200 that cannot be improved with corrective lenses, or a visualfield of less than 20 degrees diameter (10 degrees radius).

2. Sirtuin Modulators

In one aspect, the invention provides novel sirtuin-modulating compoundsfor treating and/or preventing a wide variety of diseases and disordersincluding, for example, diseases or disorders related to aging orstress, diabetes, obesity, neurodegenerative diseases, ocular diseasesand disorders, cardiovascular disease, blood clotting disorders,inflammation, cancer, and/or flushing, etc. Sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may also beused for treating a disease or disorder in a subject that would benefitfrom increased mitochondrial activity, for enhancing muscle performance,for increasing muscle ATP levels, or for treating or preventing muscletissue damage associated with hypoxia or ischemia. Other compoundsdisclosed herein may be suitable for use in a pharmaceutical compositionand/or one or more methods disclosed herein.

In one embodiment, sirtuin-modulating compounds of the invention arerepresented by Structural Formula (I):

or a salt thereof, where, as valence permits:

Ring A is optionally substituted;

L is absent, substituted or unsubstituted phenylene, substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene,substituted or unsubstituted pyrazolylene, substituted or unsubstitutedbenzothiazolylene, —NR₄—, —C(O)O—, —C(O)NR₄—, —NR₄C(O)—, —NR₄—C(O)—NR₅—,—S—, —CHR₆═CHR₇— or —CHR₆—C(O)—;

L′ is absent, substituted or unsubstituted phenylene, substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene,substituted or unsubstituted pyrazolylene, substituted or unsubstitutedbenzothiazolylene, substituted or unsubstituted indenedionylene,—C(O)O—, —C(O)NR₄—, —NR₄C(O)—, —NR₄—C(O)—NR₅—, —S—, —CHR₆═CHR₇— or—CHR₈—C(O)—, provided that at least one of L and L′ is substituted orunsubstituted phenylene, substituted or unsubstituted —O-phenylene,substituted or unsubstituted thienylene, substituted or unsubstitutedpyrazolylene, substituted or unsubstituted benzothiazolylene,substituted or unsubstituted indenedionylene, —NR₄—, —C(O)O—, —C(O)NR₄—,—NR₄C(O)—, —NR₄—C(O)—NR₅—, —S—, —CHR₆═CHR₇— or —CHR₈—C(O)—;

R₁ is absent, —H, —NR₄R₅, —N₄C(O)R₅, —OR₅, naphthyl or a heterocyclicgroup, provided that L and R₁ are not both absent unless X is N;

R₂ is —H, unsubstituted alkyl, —NR₄R₅, —NR₄C(O)R₅, —OR₅, substituted orunsubstituted phenyl, naphthyl or a heterocyclic group;

R₃ is —H, —NR₄R₅, —N₄C(O)R₅, —OR₅ or a substituted or unsubstitutedheterocyclic group, or R₂ and R₃, taken together with the atoms to whichthey are attached, form an optionally substituted heterocyclic group, orR₃ is absent when Z is O or S;

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group;

R₆, R₇ and R₈ are independently selected from the group consisting ofhalogen, —OR₄, —CN, —CO₂R₄, —OCOR₄, —OCO₂R₄, —C(O)NR₄R₅, —OC(O)NR₄R₅,—C(O)R₄, —COR₄, —SR₄, —OSO₃H, —S(O)_(n)R₄, —S(O)_(n)OR₄, —S(O)_(n)NR₄R₅,—NR₄R₅, —NR₄C(O)OR₅, —NR₄C(O)R₅ and —NO₂;

W is C or N;

X is C or N;

Y is C or N;

Z is C, N, O or S, provided that at least two of W, X, Y and Z are C;and

n is 1 or 2.

In a second embodiment, sirtuin-modulating compounds of the inventionare represented by Structural Formula (Ia):

wherein:

R₁₀ is selected from —H, —C(O)—N(R₄₀)(R₅₀), —S(O)₂N(R₄₀)(R₅₀), or—CH₂—N(R₄₀)(R₅₀); wherein each of R₄₀ and R₅₀ is independently selectedfrom —H, —C₁-C₃ straight or branched alkyl, —(C₁-C₃ straight or branchedalkyl)-N(CH₃)₂, —(C₁-C₃ straight or branched alkyl)-heterocyclyl,—(C₁-C₃ straight or branched alkyl)-alkylheterocyclyl, or wherein R₄₀and R₅₀ taken together with the N atom to which they are bound form a5-6 membered heterocyclic ring that is optionally substituted with—(C₁-C₃ straight or branched alkyl), and wherein at least one of R₄₀ orR₅₀ is not H;

R₁₁ is selected from —C₁-C₃ straight or branched alkylene or —C(O)—; and

each of ring K and ring E is independently substituted with up to threesubstituents independently selected from halo, —CF₃, —O—(C₁-C₃ straightor branched alkyl), —S—(C₁-C₃ straight or branched alkyl), —N(R₄₀)(R₅₀),—S(O)₂—N(R₄₀)(R₅₀), heterocyclyl, (C₁-C₃ straight or branchedalkyl)-heterocyclyl, —O—(C₁-C₃ straight or branched alkyl)-heterocyclyl,—S—(C₁-C₃ straight or branched alkyl)-heterocyclyl, or is optionallyfused to a 5-6 membered heterocyclyl or heteroaryl, wherein anyheterocyclyl or heteroaryl is optionally substituted with —C₁-C₃straight or branched alkyl.

In certain embodiments, one of R₄₀ or R₅₀ is H.

In certain embodiments, ring K is substituted with up to 3 substituentsindependently selected from methyl, —O-methyl, —N(CH₃)₂, or —CF₃, but isunsubstituted in the positions ortho to the attachment to the rest ofthe molecule.

In certain embodiments, such as where R₄₀ and/or R₅₀ have the valuesindicated above and/or ring K has the substitution pattern describedabove, ring E is substituted with up to 2 substituents independentlyselected from methyl, —O-methyl, —S(O)₂—N(CH₃)₂, —O-methyl-morpholino,—O-ethyl-morpholino, fluoro, —CF₃, piperidyl, methylpiperidyl,pyrrolidyl, or methylpyrrolidyl.

In certain embodiments, R₁₀ is selected from —H, —CH₂-piperazinyl,—CH₂-methylpiperazinyl, —CH₂-pyrrolidyl, —CH₂-piperidyl,—CH₂-morpholino, —CH₂—N(CH₃)₂, —C(O)—NH—(CH₂)_(n)-piperazinyl,—C(O)—NH—(CH₂)_(n)-methylpiperazinyl, —C(O)—NH—(CH₂)_(n)-pyrrolidyl,—C(O)—NH—(CH₂)_(n)-morpholino, —C(O)—NH—(CH₂)_(n)-piperidyl, or—C(O)—NH—(CH₂)_(n)N(CH₃)₂, wherein n is 1 or 2.

In particular embodiments, ring K is substituted with up to 3substituents independently selected from methyl, O-methyl, N(CH₃)₂, CF₃,but is unsubstituted in the positions ortho to the attachment to therest of the molecule; ring E is substituted with up to 2 substituentsindependently selected from methyl, O-methyl, —S(O)₂—N(CH₃)₂,—O-methyl-morpholino, —O-ethyl-morpholino, fluoro, —CF₃,methylpiperidyl, or pyrrolidyl; and R₁₀ is selected from —H,—CH₂-piperazinyl, —C(O)—NH—(CH₂)₂-piperazinyl,—C(O)—NH—(CH₂)₂-methylpiperazinyl, —C(O)—NH—(CH₂)₂-pyrrolidyl, or—C(O)—NH—(CH₂)₂—N(CH₃)₂.

In a further embodiment, sirtuin-modulating compounds of the inventionare represented by Formula (Ib):

wherein:

Z is selected from O or S;

R₁₀ is selected from —H, —C(O)—N(R₄₀)(R₅₀), —S(O)₂N(R₄₀)(R₅₀), or—CH₂—N(R₄₀)(R₅₀); wherein each of R₄₀ and R₅₀ is independently selectedfrom —H, —C₁-C₃ straight or branched alkyl, —(C₁-C₃ straight or branchedalkyl)-N(CH₃)₂, —(C₁-C₃ straight or branched alkyl)-heterocyclyl,—(C₁-C₃ straight or branched alkyl)-alkylheterocyclyl, or wherein R₄₀and R₅₀ taken together with the N atom to which they are bound form a5-6 membered heterocyclic ring that is optionally substituted with—(C₁-C₃ straight or branched alkyl), and wherein at least one of R₄₀ orR₅₀ is not H.

R₁₁ is selected from —C₁-C₃ straight or branched alkylene or —C(O)—;

each of R₁₂ and R₁₃ is independently selected from —H or —(C₁-C₃straight or branched alkyl), or R₁₂ and R₁₃ are taken together to form abenzene ring that is substituted with up to two substituentsindependently selected from —(C₁-C₃ straight or branched allyl), —CF₃ orhalo; and

ring K is substituted with up to three substituents independentlyselected from halo, —CF₃, —O—(C₁-C₃ straight or branched alkyl),—S—(C₁-C₃ straight or branched alkyl), —N(R₄₀)(R₅₀), —S(O)₂—N(R₄₀)(R₅₀),heterocyclyl, (C₁-C₃ straight or branched allyl)-heterocyclyl, —O—(C₁-C₃straight or branched alkyl)-heterocyclyl, —S—(C₁-C₃ straight or branchedalkyl)-heterocyclyl, or is optionally fused to a 5-6 memberedheterocyclyl or heteroaryl, wherein any heterocyclyl or heteroaryl isoptionally substituted with —C₁-C₃ straight or branched alkyl.

In certain embodiments, R₁₀ is —H.

In certain embodiments, ring K is substituted with up to 3 substituentsindependently selected from methyl, O-methyl, N(CH₃)₂, CF₃, and whereinring K is unsubstituted in the positions ortho to the attachment to therest of the molecule.

In certain embodiments, such as when R₁₀ is —H and/or ring K has thesubstitution pattern described above, each of R₁₂ and R₁₃ isindependently selected from —H, methyl, —O-methyl, —S(O)₂—N(CH₃)₂,—O-methyl-morpholino, —O-ethyl-morpholino, fluoro, —CF₃, piperidyl,methylpiperidyl, pyrrolidyl, or methylpyrrolidyl. In a preferredembodiment, each of R₁₂ and R₁₃ is methyl.

In another embodiment, sirtuin-modulating compounds of the invention arerepresented by Formula (II):

or a salt thereof, where:

Rings C, D and E are optionally substituted; and

x is 0 or 1.

In certain embodiments x is 0.

In certain embodiments (e.g., where x is 0), Ring C is substituted witha group that is capable of providing a trans configuration (e.g., anamide group, an optionally 2-substituted 1-alkenyl group).

One group of compounds of the invention encompassed by StructuralFormula (II) is represented by Structural Formula (III):

or a salt thereof, where:

Rings D and E are optionally substituted; and

R₄ is —H, a substituted or unsubstituted alkyl group, a substituted orunsubstituted aryl group or a substituted or unsubstituted non-aromaticheterocyclic group.

In certain embodiments, Ring E is substituted with an acylamino,heterocyclylcarbonylamino, lower alkyl or substituted or unsubstitutedalkoxy group.

In certain embodiments, Ring D is substituted with an amino group. Inparticular embodiments, Ring E is substituted with an acylamino,heterocyclylcarbonylamino, lower alkyl, or substituted or unsubstitutedalkoxy group, and Ring D is substituted with an amino group.

In certain embodiments, R₄ is a substituted alkyl group.

A group of compounds of the invention encompassed by Structural Formula(III) is represented by Structural Formula (IV):

or a salt thereof, where:

Ring E is optionally substituted; and

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group.

In certain embodiments, Ring E is substituted with an acylamino,heterocyclylcarbonylamino, lower alkyl or substituted or unsubstitutedalkoxy group.

In certain embodiments, R₄ is a substituted alkyl group. In particularembodiments, Ring E is substituted with an acylamino,heterocyclylcarbonylamino, lower alkyl or substituted or unsubstitutedalkoxy group and R₄ is a substituted alkyl group.

In certain embodiments, R₅ is a substituted or unsubstituted alkylgroup, such as an aralkyl or a cycloalkyl group (e.g., benzyl,cyclohexyl). In particular embodiments, R₅ is a substituted orunsubstituted alkyl group when Ring E is substituted with an acylamino,heterocyclylcarbonylamino, lower alkyl or substituted or unsubstitutedalkoxy group and/or R₄ is a substituted alkyl group.

One group of compounds of the invention encompassed by StructuralFormula (IV) is represented by Structural Formula (IVa):

or a salt thereof.

Another group of compounds of the invention encompassed by StructuralFormula (IV) is represented by Structural Formula (IVb):

or a salt thereof.

Yet another group of compounds of the invention encompassed byStructural Formula (IV) is represented by Structural Formula (V):

or a salt thereof, where:

R₄, R₅ and R₉ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group.

In certain embodiments, R₄ is a substituted alkyl group.

In certain embodiments, R₅ is a substituted or unsubstituted alkylgroup, such as an aralkyl or a cycloalkyl group (e.g., benzyl,cyclohexyl). In particular embodiments, R₅ is a substituted orunsubstituted alkyl group and R₄ is a substituted alkyl group.

In certain embodiments, R₉ is a C₁₋₄ alkyl group (e.g., methyl,cyclopropyl), a substituted or unsubstituted aryl group (e.g.,substituted or unsubstituted phenyl) or a substituted or unsubstitutednon-aromatic heterocyclic group (e.g., furanyl, morpholino). Inparticular embodiments, R₉ is a C₁₋₄ allyl group, a substituted orunsubstituted aryl group or a substituted or unsubstituted non-aromaticheterocyclic group when R₅ is a substituted or unsubstituted alkyl groupand/or R₄ is a substituted alkyl group

A group of compounds of the invention encompassed by Structural Formula(V) are represented by Structural Formula (VI):

or a salt thereof, where:

R₄, R₅ and R₉ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group.

In certain embodiments, R₄ is a substituted alkyl group.

In certain embodiments, R₅ is a substituted or unsubstituted alkylgroup, such as an aralkyl or a cycloalkyl group (e.g., benzyl,cyclohexyl). In particular embodiments, R₅ is a substituted orunsubstituted alkyl group and R₄ is a substituted alkyl group.

In certain embodiments, R₉ is a C₁₋₄ alkyl group (e.g., methyl,cyclopropyl), a substituted or unsubstituted aryl group (e.g.,substituted or unsubstituted phenyl) or a substituted or unsubstitutednon-aromatic heterocyclic group (e.g., furanyl, morpholino). Inparticular embodiments, R₉ is a C₁₋₄ alkyl group, a substituted orunsubstituted aryl group or a substituted or unsubstituted non-aromaticheterocyclic group when R₅ is a substituted or unsubstituted allyl groupand/or R₄ is a substituted alkyl group

In yet another embodiment, sirtuin-modulating compounds of the inventionare represented by Formula (VII):

or a salt thereof, where:

Ring F is optionally substituted;

L′ is substituted or unsubstituted phenylene, substituted orunsubstituted thienylene, substituted or unsubstituted indonedionylene,—C(O)O—, —NR₄C(O)—, —S—, —CHR₆═CHR₇— or —CHR₈—C(O)—;

R₂ is —H, unsubstituted alkyl, —NR₄R₅, —NR₄C(O)R₅, —OR₅, substituted orunsubstituted phenyl or a heterocyclic group;

R₃ is —H, —NR₄R₅, —N₄C(O)R₅, —OR₅ or a heterocyclic group, or R₂ and R₃,taken together with the atoms to which they are attached, form anoptionally substituted heterocyclic group, or R₃ is absent when Z is Oor S;

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group;

R₆, R₇ and R₈ are independently selected from the group consisting ofhalogen, —OR₄, —CN, —CO₂R₄, —OCOR₄, —OCO₂R₄, —C(O)NR₄R₅, —OC(O)NR₄R₅,—C(O)R₄, —COR₄, —SR₄, —OSO₃H, —S(O)_(n)R₄, —S(O)_(n)OR₄, —S(O)_(n)NR₄R₅,—NR₄R₅, —NR₄C(O)OR₅, —NR₄C(O)R₅ and —NO₂;

Z is C, N, O or S; and

n is 1 or 2.

In Structural Formula (VII), the dashed bond indicated as

represents a single or double bond. For the compounds represented byStructural Formula (VII), both dashed bonds cannot be double bonds, butpreferably one of the dashed bonds is a double bond and the other is asingle bond.

One group of compounds encompassed by Structural Formula (VII) arerepresented by Structural Formula (VIII):

or a salt thereof, where:

Ring G is optionally substituted;

L′ is substituted or unsubstituted phenylene, substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene,substituted or unsubstituted indonedionylene, —NR₄C(O)—, —C(O)O—, —S—,—CHR₆═CHR₇— or —CHR₈—C(O)—;

R₂ is —H, unsubstituted alkyl, —NR₄R₅, —NR₄C(O)R₅, —OR₅, substituted orunsubstituted phenyl or a heterocyclic group;

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group;

R₆, R₇ and R₈ are independently selected from the group consisting ofhalogen, —OR₄, —CN, —CO₂R₄, —OCOR₄, —OCO₂R₄, —C(O)NR₄R₅, —OC(O)NR₄R₅,—C(O)R₄, —COR₄, —SR₄, —OSO₃H, —S(O)_(n)R₄, —S(O)_(n)OR₄, —S(O)_(n)NR₄R₅,—NR₄R₅, —NR₄C(O)OR₅, —NR₄C(O)R₅ and —NO₂; and

n is 1 or 2.

In certain embodiments, L′ is substituted or unsubstituted —O-phenylene,substituted or unsubstituted thienylene or —CHR₈—C(O)—.

In certain embodiments, R₂ is —NR₄C(O)R₅ or a heterocyclic group, suchas —NR₄C(O)-substituted alkyl. In a particular embodiment, R₂ is—NR₄C(O)R₅ or a heterocyclic group and L′ is substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene or—CHR₈—C(O)—.

In certain embodiments, Ring G is unsubstituted. In a particularembodiment, Ring G is unsubstituted when R₂ is —NR₄C(O)R₅ or aheterocyclic group and/or L′ is substituted or unsubstituted—O-phenylene, substituted or unsubstituted thienylene or —CHR₈—C(O)—.

In particular embodiments, L′ is substituted or unsubstituted—O-phenylene or —CHR₈—C(O)—. In such embodiments, R₂ is preferably aheterocyclic group.

In particular embodiments, L′ is a substituted or unsubstitutedthienylene. In such embodiments, R₂ is —NR₄C(O)R₅.

Another group of compounds of the invention encompassed by StructuralFormula (VII) is represented by Structural Formula (IX):

or a salt thereof, where:

Ring H is optionally substituted;

L′ is substituted or unsubstituted phenylene, —S— or —CHR₆═CHR₇—;

R₂ is —NR₄R₅, —NR₄C(O)R₅ or a heterocyclic group;

R₃ is —H, or R₂ and R₃, taken together with the atoms to which they areattached, form an optionally substituted heterocyclic group;

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group;

R₆ and R₇ are independently selected from the group consisting ofhalogen, —OR₄, —CN, —CO₂R₄, —OCOR₄, —OCO₂R₄, —C(O)NR₄R₅, —OC(O)NR₄R₅,—C(O)R₄, —COR₄, —SR₄, —OSO₃H, —S(O)_(n)R₄, —S(O)_(n)OR₄, —S(O)_(n)NR₄R₅,—NR₄R₅, —NR₄C(O)OR₅, —NR₄C(O)R₅ and —NO₂; and

n is 1 or 2.

In certain embodiments, L′ is —S—. In particular embodiments, L′ is —S—and R₂ and R₃, taken together with the atoms to which they are attached,form an optionally substituted heterocyclic group.

In certain embodiments, L′ is a substituted or unsubstituted phenylene.In particular embodiments, L′ is a substituted or unsubstitutedphenylene and R₂ is —NR₄C(O)R₅.

In certain embodiments, L′ is —CHR₆═CHR₇—, such as —CH₂═CH₂— or—C(CN)═CH₂—. In particular embodiments, L′ is —CHR₆═CHR₇— and R₂ is—NR₄R₃ or a substituted or unsubstituted aryl group.

A further group of compounds of the invention encompassed by StructuralFormula (VII) is represented by Structural Formula (X):

or a salt thereof, where:

Ring J is optionally substituted;

L is substituted or unsubstituted phenylene, substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene,substituted or unsubstituted pyrazolylene, substituted or unsubstitutedbenzothiazolylene, —C(O)O—, —C(O)NR₄—, —NR₄C(O)—, —NR₄—C(O)—NR₅—, —S—,—CHR₆═CHR₇— or —CHR₆—C(O)—;

L′ is substituted or unsubstituted phenylene, substituted orunsubstituted —O-phenylene, substituted or unsubstituted thienylene,substituted or unsubstituted pyrazolylene, substituted or unsubstitutedbenzothiazolylene, —C(O)O—, —C(O)NR₄—, —NR₄C(O)—, —NR₄—C(O)—NR₅—, —S—,—CHR₆═CHR₇— or —CHR₈—C(O)—;

R₁ is —H, —NR₄R₅, —N₄C(O)R₅, —OR₅, naphthyl or a heterocyclic group;

R₂ is —H, unsubstituted alkyl, —NR₄R₅, —NR₄C(O)R₅, —OR₅, naphthyl or aheterocyclic group;

R₃ is —H, —NR₄R₅, —N₄C(O)R₅, —OR₅ or a substituted or unsubstitutedheterocyclic group;

R₄ and R₅ are independently —H, a substituted or unsubstituted alkylgroup, a substituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group;

R₆, R₇ and R₈ are independently selected from the group consisting ofhalogen, —OR₄, —CN, —CO₂R₄, —OCOR₄, —OCO₂R₄, —C(O)NR₄R₅, —OC(O)NR₄R₅,—C(O)R₄, —COR₄, —SR₄, —OSO₃H, —S(O)_(n)R₄, —S(O)_(n)OR₄, —S(O)_(n)NR₄R₅,—NR₄R₅, —NR₄C(O)OR₅, —NR₄C(O)R₅ and —NO₂; and

n is 1 or 2.

In certain embodiments, L is —NR₄C(O)—.

In certain embodiments, R₃ is a substituted or unsubstituted aryl group.In particular embodiments, R₃ is a substituted or unsubstituted arylgroup and L is —NR₄C(O)—.

In certain embodiments, L′ is —C(O)O—. In particular embodiments, L′ is—C(O)O— when R₃ is a substituted or unsubstituted aryl group and/or L is—NR₄C(O)—.

In certain embodiments, R₂ is an unsubstituted alkyl group. Inparticular embodiments, R₂ is an unsubstituted alkyl group when L′ is—C(O)O—, R₃ is a substituted or unsubstituted aryl group and/or L is—NR₄C(O)—.

In certain embodiments, Ring G is substituted, such as with one or more(e.g., two) alkoxy (e.g., methoxy, ethoxy) groups, for example, in thepositions ortho to the bridgehead carbon atoms. In particularembodiments, Ring G is substituted when R₂ is an unsubstituted alkylgroup, L′ is —C(O)O—, R₃ is a substituted or unsubstituted aryl groupand/or L is —NR₄C(O)—.

Novel compounds of the invention can also be used in the methodsdescribed herein.

The compounds and salts thereof described herein also include theircorresponding hydrates (e.g., hemihydrate, monohydrate, dihydrate,trihydrate, tetrahydrate) and solvates. Suitable solvents forpreparation of solvates and hydrates can generally be selected by askilled artisan.

The compounds and salts thereof can be present in amorphous orcrystalline (including co-crystalline and polymorph) forms.

Sirtuin-modulating compounds of the invention having hydroxylsubstituents, unless otherwise indicated, also include the relatedsecondary metabolites, particularly sulfate, phosphate, acyl (e.g.,acetyl, fatty acid acyl) and sugar (e.g., glucuronate, glucose)derivatives. In other words, substituent groups —OH also include —OSO₃⁻M⁺, where M⁺ is a suitable cation (preferably H⁺, NH₄ ⁺ or an alkalimetal ion such as Na⁺ or K⁺) and sugars such as

These groups are generally cleavable to —OH by hydrolysis or bymetabolic (e.g., enzymatic) cleavage.

In certain embodiments, the compounds of the invention exclude one ormore of Compounds 1-90.

Sirtuin-modulating compounds of the invention advantageously modulatethe level and/or activity of a sirtuin protein, particularly thedeacetylase activity of the sirtuin protein.

Separately or in addition to the above properties, certainsirtuin-modulating compounds of the invention do not substantially haveone or more of the following activities: inhibition of PI3-kinase,inhibition of aldoreductase, inhibition of tyrosine kinase,transactivation of EGFR tyrosine kinase, coronary dilation, orspasmolytic activity, at concentrations of the compound that areeffective for modulating the deacetylation activity of a sirtuin protein(e.g., such as a SIRT1 and/or a SIRT3 protein).

An alkyl group is a straight chained, branched or cyclic non-aromatichydrocarbon which is completely saturated. Typically, a straight chainedor branched alkyl group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10, and a cyclic alkyl group has from 3 to about 10carbon atoms, preferably from 3 to about 8. Examples of straight chainedand branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A C₁-C₄straight chained or branched alkyl group is also referred to as a “loweralkyl” group.

An alkenyl group is a straight chained, branched or cyclic non-aromatichydrocarbon which contains one or more double bonds. Typically, thedouble bonds are not located at the terminus of the alkenyl group, suchthat the double bond is not adjacent to another functional group.

An alkynyl group is a straight chained, branched or cyclic non-aromatichydrocarbon which contains one or more triple bonds. Typically, thetriple bonds are not located at the terminus of the alkynyl group, suchthat the triple bond is not adjacent to another functional group.

A cyclic group includes carbocyclic and heterocyclic rings. Such ringscan be saturated or unsaturated, including aromatic. Heterocyclic ringstypically contain 1 to 4 heteroatoms, although oxygen and sulfur atomscannot be adjacent to each other.

Aromatic (aryl) groups include carbocyclic aromatic groups such asphenyl, naphthyl, and anthracyl, and heteroaryl groups such asimidazolyl, thienyl, furanyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl,pyrroyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is fused to one ormore other heteroaryl rings. Examples include benzothienyl,benzofuranyl, indolyl, quinolinyl, benzothiazole, benzooxazole,benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.

Non-aromatic heterocyclic rings are non-aromatic carbocyclic rings whichinclude one or more heteroatoms such as nitrogen, oxygen or sulfur inthe ring. The ring can be five, six, seven or eight-membered. Examplesinclude tetrahydrofuranyl, tetrahydrothiophenyl, morpholino,thiomorpholino, pyrrolidinyl, piperazinyl, piperidinyl, andthiazolidinyl, along with the cyclic form of sugars.

A ring fused to a second ring shares at least one common bond.

Suitable substituents on an alkyl, alkenyl, alkynyl, aryl, non-aromaticheterocyclic or aryl group (carbocyclic and heteroaryl) are those whichdo not substantially interfere with the ability of the disclosedcompounds to have one or more of the properties disclosed herein. Asubstituent substantially interferes with the properties of a compoundwhen the magnitude of the property is reduced by more than about 50% ina compound with the substituent compared with a compound without thesubstituent. Examples of suitable substituents include —OH, halogen(—Br, —Cl, —I and —F), —OR^(a), —O—COR^(a), —COR^(a), —C(O)R^(a), —CN,—NO², —COOH, —COOR^(a), —OCO₂R^(a), —C(O)NR^(a)R^(b), —OC(O)NR^(a)R^(b),—SO₃H, —NH₂, —NHR^(a), —N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂,—CONHR^(a), —CON(R^(a)R^(b)), —NHCOR^(a), —NRCOR^(a), —NHCONH₂,—NHCONR^(a)H, —NHCON(R^(a)R^(b)), —NR^(c)CONH₂, —NR^(c)CONR^(a)H,—NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂, —C(═NH)—NHR^(a),—C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂, —C(═NR^(c))—NHR^(a),—C(═NR^(c))—N(R^(a)R^(b)), —NH—C(═NH)—NH₂, —NH—C(═NH)—NHR^(a),—NH—C(═NH)—N(R^(a)R^(b)), —NH—C(═NR^(c))—NH₂, —NH—C(═NR^(c))—NHR^(a),—NH—C(═NR^(c))—N(R^(a)R^(b)), —NR^(d)H—C(═NH)—NH₂,—NR^(d)—C(═NH)—NHR^(a), —NR^(d)—C(═NH)—N(R^(a)R^(b)),—NR^(d)—C(═NR^(c))—NH₂, —NR^(d)—C(═NR^(c))—NHR^(a),—NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂, —NHNHR^(a), —NHR^(a)R^(b),—SO₂NH₂, —SO₂NHR_(a), —SO₂NR^(a)R^(b), —CH═CHR^(a), —CH═CR^(a)R^(b),—CR^(c)═CR^(a)R^(b), CR^(c)═CHR^(a), —CR^(c)═CR^(a)R^(b), —CCR^(a), —SH,—SO_(k)R^(a) (k is 0, 1 or 2), —S(O)_(k)OR^(a) (k is 0, 1 or 2) and—NH—C(═NH)—NH₂. R^(a)-R^(d) are each independently an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aromatic orsubstituted aromatic group, preferably an alkyl, benzylic or aryl group.In addition, —NR^(a)R^(b), taken together, can also form a substitutedor unsubstituted non-aromatic heterocyclic group. A non-aromaticheterocyclic group, benzylic group or aryl group can also have analiphatic or substituted aliphatic group as a substituent. A substitutedaliphatic group can also have a non-aromatic heterocyclic ring, asubstituted a non-aromatic heterocyclic ring, benzyl, substitutedbenzyl, aryl or substituted aryl group as a substituent. A substitutedaliphatic, non-aromatic heterocyclic group, substituted aryl, orsubstituted benzyl group can have more than one substituent.

A hydrogen-bond donating group is a functional group having a partiallypositively-charged hydrogen atom (e.g., —OH, —NH₂, —SH) or a group(e.g., an ester) that metabolizes into a group capable of donating ahydrogen bond.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. As usedherein, the term “stable” refers to compounds that possess stabilitysufficient to allow manufacture and that maintain the integrity of thecompound for a sufficient period of time to be useful for the purposesdetailed herein.

Double bonds indicated in a structure as:

are intended to include both the (E)- and (Z)-configuration. Preferably,double bonds are in the (E)-configuration.

A sugar is an aldehyde or ketone derivative of a straight-chainpolyhydroxy alcohol, which contains at least three carbon atoms. A sugarcan exist as a linear molecule or, preferably, as a cyclic molecule(e.g., in the pyranose or furanose form). Preferably, a sugar is amonosaccharide such as glucose or glucuronic acid. In embodiments of theinvention where, for example, prolonged residence of a compoundderivatized with a sugar is desired, the sugar is preferably anon-naturally occurring sugar. For example, one or more hydroxyl groupsare substituted with another group, such as a halogen (e.g., chlorine).The stereochemical configuration at one or more carbon atoms can also bealtered, as compared to a naturally occurring sugar. One example of asuitable non-naturally occurring sugar is sucralose.

A fatty acid is a carboxylic acid having a long-chained hydrocarbonmoiety. Typically, a fatty acid has an even number of carbon atomsranging from 12 to 24, often from 14 to 20. Fatty acids can be saturatedor unsaturated and substituted or unsubstituted, but are typicallyunsubstituted. Fatty acids can be naturally or non-naturally occurring.In embodiments of the invention where, for example, prolonged residencetime of a compound having a fatty acid moiety is desired, the fatty acidis preferably non-naturally occurring. The acyl group of a fatty acidconsists of the hydrocarbon moiety and the carbonyl moiety of thecarboxylic acid functionality, but excludes the —OH moiety associatedwith the carboxylic acid functionality.

Also included in the present invention are salts, particularlypharmaceutically acceptable salts, of the sirtuin-modulating compoundsdescribed herein. The compounds of the present invention that possess asufficiently acidic, a sufficiently basic, or both functional groups,can react with any of a number of inorganic bases, and inorganic andorganic acids, to form a salt. Alternatively, compounds that areinherently charged, such as those with a quaternary nitrogen, can form asalt with an appropriate counterion (e.g., a halide such as bromide,chloride, or fluoride, particularly bromide).

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

In an exemplary embodiment, a sirtuin-modulating compound may traversethe cytoplasmic membrane of a cell. For example, a compound may have acell-permeability of at least about 20%, 50%, 75%, 80%, 90% or 95%.

Sirtuin-modulating compounds described herein may also have one or moreof the following characteristics: the compound may be essentiallynon-toxic to a cell or subject; the sirtuin-modulating compound may bean organic molecule or a small molecule of 2000 amu or less, 1000 amu orless; a compound may have a half-life under normal atmosphericconditions of at least about 30 days, 60 days, 120 days, 6 months or 1year; the compound may have a half-life in solution of at least about 30days, 60 days, 120 days, 6 months or 1 year; a sirtuin-modulatingcompound may be more stable in solution than resveratrol by at least afactor of about 50%, 2 fold, 5 fold, 10 fold, 30 fold, 50 fold or 100fold; a sirtuin-modulating compound may promote deacetylation of the DNArepair factor Ku70; a sirtuin-modulating compound may promotedeacetylation of RelA/p65; a compound may increase general turnoverrates and enhance the sensitivity of cells to TNF-induced apoptosis.

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to inhibit a histone deacetylase (HDACs) class I, aHDAC class II, or HDACs I and II, at concentrations (e.g., in vivo)effective for modulating the deacetylase activity of the sirtuin. Forinstance, in preferred embodiments the sirtuin-modulating compound is asirtuin-activating compound and is chosen to have an EC₅₀ for activatingsirtuin deacetylase activity that is at least 5 fold less than the EC₅₀for inhibition of an HDAC I and/or HDAC II, and even more preferably atleast 10 fold, 100 fold or even 1000 fold less. Methods for assayingHDAC I and/or HDAC II activity are well known in the art and kits toperform such assays may be purchased commercially. See e.g., BioVision,Inc. (Mountain View, Calif.; world wide web at biovision.com) and ThomasScientific (Swedesboro, N.J.; world wide web at tomassci.com).

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to modulate sirtuin homologs. In one embodiment, anactivator of a human sirtuin protein may not have any substantialability to activate a sirtuin protein from lower eukaryotes,particularly yeast or human pathogens, at concentrations (e.g., in vivo)effective for activating the deacetylase activity of human sirtuin. Forexample, a sirtuin-activating compound may be chosen to have an EC₅₀ foractivating a human sirtuin, such as SIRT1 and/or SIRT3, deacetylaseactivity that is at least 5 fold less than the EC₅₀ for activating ayeast sirtuin, such as Sir2 (such as Candida, S. cerevisiae, etc.), andeven more preferably at least 10 fold, 100 fold or even 1000 fold less.In another embodiment, an inhibitor of a sirtuin protein from lowereukaryotes, particularly yeast or human pathogens, does not have anysubstantial ability to inhibit a sirtuin protein from humans atconcentrations (e.g., in vivo) effective for inhibiting the deacetylaseactivity of a sirtuin protein from a lower eukaryote. For example, asirtuin-inhibiting compound may be chosen to have an IC₅₀ for inhibitinga human sirtuin, such as SIRT1 and/or SIRT3, deacetylase activity thatis at least 5 fold less than the IC₅₀ for inhibiting a yeast sirtuin,such as Sir2 (such as Candida, S. cerevisiae, etc.), and even morepreferably at least 10 fold, 100 fold or even 1000 fold less.

In certain embodiments, a sirtuin-modulating compound may have theability to modulate one or more sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,or SIRT7. In one embodiment, a sirtuin-modulating compound has theability to modulate both a SIRT1 and a SIRT3 protein.

In other embodiments, a SIRT1 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT1, For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT1deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In one embodiment, a SIRT1 modulator does not have anysubstantial ability to modulate a SIRT3 protein.

In other embodiments, a SIRT3 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT3. For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT3deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In one embodiment, a SIRT3 modulator does not have anysubstantial ability to modulate a SIRT1 protein.

In certain embodiments, a sirtuin-modulating compound may have a bindingaffinity for a sirtuin protein of about 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, 10⁻¹²M orless. A sirtuin-modulating compound may reduce (activator) or increase(inhibitor) the apparent Km of a sirtuin protein for its substrate orNAD+ (or other cofactor) by a factor of at least about 2, 3, 4, 5, 10,20, 30, 50 or 100. In certain embodiments, Km values are determinedusing the mass spectrometry assay described herein. Preferred activatorcompounds reduce the Km of a sirtuin for its substrate or cofactor to agreater extent than caused by resveratrol at a similar concentration orreduce the Km of a sirtuin for its substrate or cofactor similar to thatcaused by resveratrol at a lower concentration. A sirtuin-modulatingcompound may increase the Vmax of a sirtuin protein by a factor of atleast about 2, 3, 4, 5, 10, 20, 30, 50 or 100. A sirtuin-modulatingcompound may have an ED50 for modulating the deacetylase activity of aSIRT1 and/or SIRT3 protein of less than about 1 nM, less than about 10nM, less than about 100 nM, less than about 1 μM, less than about 10 μM,less than about 100 μM, or from about 1-10 nM, from about 10-100 nM,from about 0.1-1 μM, from about 1-10 μM or from about 10-100 μM. Asirtuin-modulating compound may modulate the deacetylase activity of aSIRT1 and/or SIRT3 protein by a factor of at least about 5, 10, 20, 30,50, or 100, as measured in a cellular assay or in a cell based assay. Asirtuin-activating compound may cause at least about 10%, 30%, 50%, 80%,2 fold, 5 fold, 10 fold, 50 fold or 100 fold greater induction of thedeacetylase activity of a sirtuin protein relative to the sameconcentration of resveratrol. A sirtuin-modulating compound may have anED50 for modulating SIRT5 that is at least about 10 fold, 20 fold, 30fold, 50 fold greater than that for modulating SIRT1 and/or SIRT3.

3. Exemplary Uses

In certain aspects, the invention provides methods for modulating thelevel and/or activity of a sirtuin protein and methods of use thereof.

In certain embodiments, the invention provides methods for usingsirtuin-modulating compounds wherein the sirtuin-modulating compoundsactivate a sirtuin protein, e.g., increase the level and/or activity ofa sirtuin protein. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for a variety oftherapeutic applications including, for example, increasing the lifespanof a cell, and treating and/or preventing a wide variety of diseases anddisorders including, for example, diseases or disorders related to agingor stress, diabetes, obesity, neurodegenerative diseases, cardiovasculardisease, blood clotting disorders, inflammation, cancer, and/orflushing, etc. The methods comprise administering to a subject in needthereof a pharmaceutically effective amount of a sirtuin-modulatingcompound, e.g., a sirtuin-activating compound.

In other embodiments, the invention provides methods for usingsirtuin-modulating compounds wherein the sirtuin-modulating compoundsdecrease sirtuin activity, e.g., decrease the level and/or activity of asirtuin protein. Sirtuin-modulating compounds that decrease the leveland/or activity of a sirtuin protein may be useful for a variety oftherapeutic application including, for example, increasing cellularsensitivity to stress (including increasing radiosensitivity and/orchemosensitivity), increasing the amount and/or rate of apoptosis,treatment of cancer (optionally in combination another chemotherapeuticagent), stimulation of appetite, and/or stimulation of weight gain, etc.The methods comprise administering to a subject in need thereof apharmaceutically effective amount of a sirtuin-modulating compound,e.g., a sirtuin-inhibiting compound.

While Applicants do not wish to be bound by theory, it is believed thatactivators and inhibitors of the instant invention may interact with asirtuin at the same location within the sirtuin protein (e.g., activesite or site affecting the Kin or Vmax of the active site). It isbelieved that this is the reason why certain classes of sirtuinactivators and inhibitors can have substantial structural similarity.

In certain embodiments, the sirtuin-modulating compounds describedherein may be taken alone or in combination with other compounds. In oneembodiment, a mixture of two or more sirtuin-modulating compounds may beadministered to a subject in need thereof. In another embodiment, asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein may be administered with one or more of the followingcompounds: resveratrol, butein, fisetin, piceatannol, or quercetin. Inan exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered incombination with nicotinic acid. In another embodiment, asirtuin-modulating compound that decreases the level and/or activity ofa sirtuin protein may be administered with one or more of the followingcompounds: nicotinamide (NAM), suranim; NF023 (a G-protein antagonist);NF279 (a purinergic receptor antagonist); Trolox(6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid);(−)-epigallocatechin (hydroxy on sites 3,5,7,3′,4′,5′);(−)-epigallocatechin gallate (Hydroxy sites 5,7,3′,4′,5′ and gallateester on 3); cyanidin chloride (3,5,7,3′,4′-pentahydroxyflavyliumchloride); delphinidin chloride (3,5,7,3′,4′,5′-hexahydroxyflavyliumchloride); myricetin (cannabiscetin; 3,5,7,3′,4′,5′-hexahydroxyflavone);3,7,3′,4′,5′-pentahydroxyflavone; gossypetin(3,5,7,8,3′,4′-hexahydroxyflavone), sirtinol; and splitomicin (see e.g.,Howitz et al. (2003) Nature 425:191; Grozinger et al. (2001) J. Biol.Chem. 276:38837; Dedalov et al. (2001) PNAS 98:15113; and Hirao et al.(2003) J. Biol. Chem 278:52773). In yet another embodiment, one or moresirtuin-modulating compounds may be administered with one or moretherapeutic agents for the treatment or prevention of various diseases,including, for example, cancer, diabetes, neurodegenerative diseases,cardiovascular disease, blood clotting, inflammation, flushing, obesity,ageing, stress, etc. In various embodiments, combination therapiescomprising a sirtuin-modulating compound may refer to (1) pharmaceuticalcompositions that comprise one or more sirtuin-modulating compounds incombination with one or more therapeutic agents (e.g., one or moretherapeutic agents described herein); and (2) co-administration of oneor more sirtuin-modulating compounds with one or more therapeutic agentswherein the sirtuin-modulating compound and therapeutic agent have notbeen formulated in the same compositions (but may be present within thesame kit or package, such as a blister pack or other multi-chamberpackage; connected, separately sealed containers (e.g., foil pouches)that can be separated by the user; or a kit where the sirtuin-modulatingcompound(s) and other therapeutic agent(s) are in separate vessels).When using separate formulations, the sirtuin-modulating compound may beadministered at the same, intermittent, staggered, prior to, subsequentto, or combinations thereof, with the administration of anothertherapeutic agent.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a sirtuin-modulating compound may alsocomprise increasing the protein level of a sirtuin, such as human SIRT1and/or SIRT3, or homologs thereof. Increasing protein levels can beachieved by introducing into a cell one or more copies of a nucleic acidthat encodes a sirtuin. For example, the level of a sirtuin can beincreased in a mammalian cell by introducing into the mammalian cell anucleic acid encoding the sirtuin, e.g., increasing the level of SIRT1by introducing a nucleic acid encoding the amino acid sequence set forthin GenBank Accession No. NP_(—)036370 and/or increasing the level ofSIRT3 by introducing a nucleic acid encoding the amino acid sequence setforth in GenBank Accession No. AAH01042. The nucleic acid may be underthe control of a promoter that regulates the expression of the SIRT1and/or SIRT3 nucleic acid. Alternatively, the nucleic acid may beintroduced into the cell at a location in the genome that is downstreamof a promoter. Methods for increasing the level of a protein using thesemethods are well known in the art.

A nucleic acid that is introduced into a cell to increase the proteinlevel of a sirtuin may encode a protein that is at least about 80%, 85%,90%, 95%, 98%, or 99% identical to the sequence of a sirtuin, e.g.,SIRT1 (GenBank Accession No. NP_(—)036370) and/or SIRT3 (GenBankAccession No. AAH01042) protein. For example, the nucleic acid encodingthe protein may be at least about 80%, 85%, 90%, 95%, 98%, or 99%identical to a nucleic acid encoding a SIRT1 (e.g. GenBank Accession No.NM_(—)012238) and/or SIRT3 (e.g., GenBank Accession No. BC001042)protein. The nucleic acid may also be a nucleic acid that hybridizes,preferably under stringent hybridization conditions, to a nucleic acidencoding a wild-type sirtuin, e.g., SIRT1 (GenBank Accession No.NM_(—)012238) and/or SIRT3 (e.g., GenBank Accession No. BC001042)protein. Stringent hybridization conditions may include hybridizationand a wash in 0.2×SSC at 65° C. When using a nucleic acid that encodes aprotein that is different from a wild-type sirtuin protein, such as aprotein that is a fragment of a wild-type sirtuin, the protein ispreferably biologically active, e.g., is capable of deacetylation. It isonly necessary to express in a cell a portion of the sirtuin that isbiologically active. For example, a protein that differs from wild-typeSIRT1 having GenBank Accession No. NP_(—)036370, preferably contains thecore structure thereof. The core structure sometimes refers to aminoacids 62-293 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 237 to 932 of GenBank Accession No. NM_(—)012238, whichencompasses the NAD binding as well as the substrate binding domains.The core domain of SIRT1 may also refer to about amino acids 261 to 447of GenBank Accession No. NP_(—)036370, which are encoded by nucleotides834 to 1394 of GenBank Accession No. NM_(—)012238; to about amino acids242 to 493 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 777 to 1532 of GenBank Accession No. NM_(—)012238; or toabout amino acids 254 to 495 of GenBank Accession No. NP_(—)036370,which are encoded by nucleotides 813 to 1538 of GenBank Accession No.NM_(—)012238. Whether a protein retains a biological function, e.g.,deacetylation capabilities, can be determined according to methods knownin the art.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a sirtuin-modulating compound may alsocomprise decreasing the protein level of a sirtuin, such as human SIRT1and/or SIRT3, or homologs thereof. Decreasing a sirtuin protein levelcan be achieved according to methods known in the art. For example, ansiRNA, an antisense nucleic acid, or a ribozyme targeted to the sirtuincan be expressed in the cell. A dominant negative sirtuin mutant, e.g.,a mutant that is not capable of deacetylating, may also be used. Forexample, mutant H363Y of SIRT1, described, e.g., in Luo et al. (2001)Cell 107:137 can be used. Alternatively, agents that inhibittranscription can be used.

Methods for modulating sirtuin protein levels also include methods formodulating the transcription of genes encoding sirtuins, methods forstabilizing/destabilizing the corresponding mRNAs, and other methodsknown in the art.

Aging/Stress

In one embodiment, the invention provides a method extending thelifespan of a cell, extending the proliferative capacity of a cell,slowing ageing of a cell, promoting the survival of a cell, delayingcellular senescence in a cell, mimicking the effects of calorierestriction, increasing the resistance of a cell to stress, orpreventing apoptosis of a cell, by contacting the cell with asirtuin-modulating compound of the invention that increases the leveland/or activity of a sirtuin protein. In an exemplary embodiment, themethods comprise contacting the cell with a sirtuin-activating compound.

The methods described herein may be used to increase the amount of timethat cells, particularly primary cells (i.e., cells obtained from anorganism, e.g., a human), may be kept alive in a cell culture. Embryonicstem (ES) cells and pluripotent cells, and cells differentiatedtherefrom, may also be treated with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein to keep thecells, or progeny thereof, in culture for longer periods of time. Suchcells can also be used for transplantation into a subject, e.g., afterex vivo modification.

In one embodiment, cells that are intended to be preserved for longperiods of time may be treated with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein. The cells maybe in suspension (e.g., blood cells, serum, biological growth media,etc.) or in tissues or organs. For example, blood collected from anindividual for purposes of transfusion may be treated with asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein to preserve the blood cells for longer periods oftime. Additionally, blood to be used for forensic purposes may also bepreserved using a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein. Other cells that may be treated toextend their lifespan or protect against apoptosis include cells forconsumption, e.g., cells from non-human mammals (such as meat) or plantcells (such as vegetables).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be applied during developmental and growthphases in mammals, plants, insects or microorganisms, in order to, e.g.,alter, retard or accelerate the developmental and/or growth process.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to treat cellsuseful for transplantation or cell therapy, including, for example,solid tissue grafts, organ transplants, cell suspensions, stem cells,bone marrow cells, etc. The cells or tissue may be an autograft, anallograft, a syngraft or a xenograft. The cells or tissue may be treatedwith the sirtuin-modulating compound prior toadministration/implantation, concurrently withadministration/implantation, and/or post administration/implantationinto a subject. The cells or tissue may be treated prior to removal ofthe cells from the donor individual, ex vivo after removal of the cellsor tissue from the donor individual, or post implantation into therecipient. For example, the donor or recipient individual may be treatedsystemically with a sirtuin-modulating compound or may have a subset ofcells/tissue treated locally with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein. In certainembodiments, the cells or tissue (or donor/recipient individuals) mayadditionally be treated with another therapeutic agent useful forprolonging graft survival, such as, for example, an immunosuppressiveagent, a cytokine, an angiogenic factor, etc.

In yet other embodiments, cells may be treated with a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin proteinin vivo, e.g., to increase their lifespan or prevent apoptosis. Forexample, skin can be protected from aging (e.g., developing wrinkles,loss of elasticity, etc.) by treating skin or epithelial cells with asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein. In an exemplary embodiment, skin is contacted with apharmaceutical or cosmetic composition comprising a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.Exemplary skin afflictions or skin conditions that may be treated inaccordance with the methods described herein include disorders ordiseases associated with or caused by inflammation, sun damage ornatural aging. For example, the compositions find utility in theprevention or treatment of contact dermatitis (including irritantcontact dermatitis and allergic contact dermatitis), atopic dermatitis(also known as allergic eczema), actinic keratosis, keratinizationdisorders (including eczema), epidermolysis bullosa diseases (includingpenfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas(including erythema multiforme and erythema nodosum), damage caused bythe sun or other light sources, discoid lupus erythematosus,dermatomyositis, psoriasis, skin cancer and the effects of naturalaging. In another embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be used for thetreatment of wounds and/or burns to promote healing, including, forexample, first-, second- or third-degree burns and/or a thermal,chemical or electrical burns. The formulations may be administeredtopically, to the skin or mucosal tissue, as an ointment, lotion, cream,microemulsion, gel, solution or the like, as further described herein,within the context of a dosing regimen effective to bring about thedesired result.

Topical formulations comprising one or more sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may also beused as preventive, e.g., chemopreventive, compositions. When used in achemopreventive method, susceptible skin is treated prior to any visiblecondition in a particular individual.

Sirtuin-modulating compounds may be delivered locally or systemically toa subject. In one embodiment, a sirtuin-modulating compound is deliveredlocally to a tissue or organ of a subject by injection, topicalformulation, etc.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used for treating orpreventing a disease or condition induced or exacerbated by cellularsenescence in a subject; methods for decreasing the rate of senescenceof a subject, e.g., after onset of senescence; methods for extending thelifespan of a subject; methods for treating or preventing a disease orcondition relating to lifespan; methods for treating or preventing adisease or condition relating to the proliferative capacity of cells;and methods for treating or preventing a disease or condition resultingfrom cell damage or death. In certain embodiments, the method does notact by decreasing the rate of occurrence of diseases that shorten thelifespan of a subject. In certain embodiments, a method does not act byreducing the lethality caused by a disease, such as cancer.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered to asubject in order to generally increase the lifespan of its cells and toprotect its cells against stress and/or against apoptosis. It isbelieved that treating a subject with a compound described herein issimilar to subjecting the subject to hormesis, i.e., mild stress that isbeneficial to organisms and may extend their lifespan.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to a subject to prevent aging andaging-related consequences or diseases, such as stroke, heart disease,heart failure, arthritis, high blood pressure, and Alzheimer's disease.Other conditions that can be treated include ocular disorders, e.g.,associated with the aging of the eye, such as cataracts, glaucoma, andmacular degeneration. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can also be administered tosubjects for treatment of diseases, e.g., chronic diseases, associatedwith cell death, in order to protect the cells from cell death.Exemplary diseases include those associated with neural cell death,neuronal dysfunction, or muscular cell death or dysfunction, such asParkinson's disease, Alzheimer's disease, multiple sclerosis,amniotropic lateral sclerosis, and muscular dystrophy; AIDS; fulminanthepatitis; diseases linked to degeneration of the brain, such asCreutzfeld-Jakob disease, retinitis pigmentosa and cerebellardegeneration; myelodysplasis such as aplastic anemia; ischemic diseasessuch as myocardial infarction and stroke; hepatic diseases such asalcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such asosteoarthritis; atherosclerosis; alopecia; damage to the skin due to UVlight; lichen planus; atrophy of the skin; cataract; and graftrejections. Cell death can also be caused by surgery, drug therapy,chemical exposure or radiation exposure.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein can also be administered to a subject suffering froman acute disease, e.g., damage to an organ or tissue, e.g., a subjectsuffering from stroke or myocardial infarction or a subject sufferingfrom a spinal cord injury. Sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may also be used torepair an alcoholic's liver.

Cardiovascular Disease

In another embodiment, the invention provides a method for treatingand/or preventing a cardiovascular disease by administering to a subjectin need thereof a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein.

Cardiovascular diseases that can be treated or prevented using thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein include cardiomyopathy or myocarditis; such asidiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholiccardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy,and hypertensive cardiomyopathy. Also treatable or preventable usingcompounds and methods described herein are atheromatous disorders of themajor blood vessels (macrovascular disease) such as the aorta, thecoronary arteries, the carotid arteries, the cerebrovascular arteries,the renal arteries, the iliac arteries, the femoral arteries, and thepopliteal arteries. Other vascular diseases that can be treated orprevented include those related to platelet aggregation, the retinalarterioles, the glomerular arterioles, the vasa nervorum, cardiacarterioles, and associated capillary beds of the eye, the kidney, theheart, and the central and peripheral nervous systems. Thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used for increasing HDL levels in plasmaof an individual.

Yet other disorders that may be treated with sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteininclude restenosis, e.g., following coronary intervention, and disordersrelating to an abnormal level of high density and low densitycholesterol.

In one embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be administered as partof a combination therapeutic with another cardiovascular agentincluding, for example, an anti-arrhythmic agent, an antihypertensiveagent, a calcium channel blocker, a cardioplegic solution, a cardiotonicagent, a fibrinolytic agent, a sclerosing solution, a vasoconstrictoragent, a vasodilator agent, a nitric oxide donor, a potassium channelblocker, a sodium channel blocker, statins, or a naturiuretic agent.

In one embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be administered as partof a combination therapeutic with an anti-arrhythmia agent.Anti-arrhythmia agents are often organized into four main groupsaccording to their mechanism of action: type I, sodium channel blockade;type II, beta-adrenergic blockade; type I, repolarization prolongation;and type IV, calcium channel blockade. Type I anti-arrhythmic agentsinclude lidocaine, moricizine, mexiletine, tocainide, procainamide,encainide, flecanide, tocainide, phenyloin, propafenone, quinidine,disopyramide, and flecainide. Type II anti-arrhythmic agents includepropranolol and esmolol. Type III includes agents that act by prolongingthe duration of the action potential, such as amiodarone, artilide,bretylium, clofilium, isobutilide, sotalol, azimilide, dofetilide,dronedarone, ersentilide, ibutilide, tedisamil, and trecetilide. Type IVanti-arrhythmic agents include verapamil, diltaizem, digitalis,adenosine, nickel chloride, and magnesium ions.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be administered as partof a combination therapeutic with another cardiovascular agent. Examplesof cardiovascular agents include vasodilators, for example, hydralazine;angiotensin converting enzyme inhibitors, for example, captopril;anti-anginal agents, for example, isosorbide nitrate, glyceryltrinitrate and pentaerythritol tetranitrate; anti-arrhythmic agents, forexample, quinidine, procainaltide and lignocaine; cardioglycosides, forexample, digoxin and digitoxin; calcium antagonists, for example,verapamil and nifedipine; diuretics, such as thiazides and relatedcompounds, for example, bendrofluazide, chlorothiazide, chlorothalidone,hydrochlorothiazide and other diuretics, for example, fursemide andtriamterene, and sedatives, for example, nitrazepam, flurazepam anddiazepam.

Other exemplary cardiovascular agents include, for example, acyclooxygenase inhibitor such as aspirin or indomethacin, a plateletaggregation inhibitor such as clopidogrel, ticlopidene or aspirin,fibrinogen antagonists or a diuretic such as chlorothiazide,hydrochlorothiazide, flumethiazide, hydroflumethiazide,bendroflumethiazide, methylchlorthiazide, trichloromethiazide,polythiazide or benzthiazide as well as ethacrynic acid tricrynafen,chlorthalidone, furosemide, musolimine, bumetanide, triamterene,amiloride and spironolactone and salts of such compounds, angiotensinconverting enzyme inhibitors such as captopril, zofenopril, fosinopril,enalapril, ceranopril, cilazopril, delapril, pentopril, quinapril,ramipril, lisinopril, and salts of such compounds, angiotensin IIantagonists such as losartan, irbesartan or valsartan, thrombolyticagents such as tissue plasminogen activator (tPA), recombinant tPA,streptokinase, urokinase, prourokinase, and anisoylated plasminogenstreptokinase activator complex (APSAC, Eminase, Beecham Laboratories),or animal salivary gland plasminogen activators, calcium channelblocking agents such as verapamil, nifedipine or diltiazem, thromboxanereceptor antagonists such as ifetroban, prostacyclin mimetics, orphosphodiesterase inhibitors. Such combination products if formulated asa fixed dose employ the compounds of this invention within the doserange described above and the other pharmaceutically active agent withinits approved dose range.

Yet other exemplary cardiovascular agents include, for example,vasodilators, e.g., bencyclane, cinnarizine, citicoline, cyclandelate,cyclonicate, ebumamonine, phenoxezyl, flunarizine, ibudilast,ifenprodil, lomerizine, naphlole, nikamate, nosergoline, nimodipine,papaverine, pentifylline, nofedoline, vincamin, vinpocetine, vichizyl,pentoxifylline, prostacyclin derivatives (such as prostaglandin E1 andprostaglandin I2), an endothelin receptor blocking drug (such asbosentan), diltiazem, nicorandil, and nitroglycerin. Examples of thecerebral protecting drug include radical scavengers (such as edaravone,vitamin E, and vitamin C), glutamate antagonists, AMPA antagonists,kainate antagonists, NMDA antagonists, GABA agonists, growth factors,opioid antagonists, phosphatidylcholine precursors, serotonin agonists,Na⁺/Ca²⁺ channel inhibitory drugs, and K⁺ channel opening drugs.Examples of the brain metabolic stimulants include amantadine, tiapride,and .gamma.-aminobutyric acid. Examples of the anticoagulant includeheparins (such as heparin sodium, heparin potassium, dalteparin sodium,dalteparin calcium, heparin calcium, parnaparin sodium, reviparinsodium, and danaparoid sodium), warfarin, enoxaparin, argatroban,batroxobin, and sodium citrate. Examples of the antiplatelet druginclude ticlopidine hydrochloride, dipyridamole, cilostazol, ethylicosapentate, sarpogrelate hydrochloride, dilazep hydrochloride,trapidil, a nonsteroidal antiinflammatory agent (such as aspirin),beraprostsodium, iloprost, and indobufene. Examples of the thrombolyticdrug include urokinase, tissue-type plasminogen activators (such asalteplase, tisokinase, nateplase, pamiteplase, monteplase, andrateplase), and nasaruplase. Examples of the antihypertensive druginclude angiotensin converting enzyme inhibitors (such as captopril,alacepril, lisinopril, imidapril, quinapril, temocapril, delapril,benazepril, cilazapril, trandolapril, enalapril, ceronapril, fosinopril,imadapril, mobertpril, perindopril, ramipril, spirapril, andrandolapril), angiotensin II antagonists (such as losartan, candesartan,valsartan, eprosartan, and irbesartan), calcium channel blocking drugs(such as aranidipine, efonidipine, nicardipine, bamidipine, benidipine,manidipine, cilnidipine, nisoldipine, nitrendipine, nifedipine,nilvadipine, felodipine, amlodipine, diltiazem, bepridil, clentiazem,phendilin, galopamil, mibefradil, prenylamine, semotiadil, terodiline,verapamil, cilnidipine, elgodipine, isradipine, lacidipine,lercanidipine, nimodipine, cinnarizine, flunarizine, lidoflazine,lomerizine, bencyclane, etafenone, and perhexyline), β-adrenalinereceptor blocking drugs (propranolol, pindolol, indenolol, carteolol,bunitrolol, atenolol, acebutolol, metoprolol, timolol, nipradilol,penbutolol, nadolol, tilisolol, carvedilol, bisoprolol, betaxolol,celiprolol, bopindolol, bevantolol, labetalol, alprenolol, amosulalol,arotinolol, befunolol, bucumolol, bufetolol, buferalol, buprandolol,butylidine, butofilolol, carazolol, cetamolol, cloranolol, dilevalol,epanolol, levobunolol, mepindolol, metipranolol, moprolol, nadoxolol,nevibolol, oxprenolol, practol, pronetalol, sotalol, sufinalol,talindolol, tertalol, toliprolol, xybenolol, and esmolol), α-receptorblocking drugs (such as amosulalol, prazosin, terazosin, doxazosin,bunazosin, urapidil, phentolamine, arotinolol, dapiprazole, fenspiride,indoramin, labetalol, naftopidil, nicergoline, tamsulosin, tolazoline,trimazosin, and yohimbine), sympathetic nerve inhibitors (such asclonidine, guanfacine, guanabenz, methyldopa, and reserpine),hydralazine, todralazine, budralazine, and cadralazine. Examples of theantianginal drug include nitrate drugs (such as amyl nitrite,nitroglycerin, and isosorbide), β-adrenaline receptor blocking drugs(such as propranolol, pindolol, indenolol, carteolol, bunitrolol,atenolol, acebutolol, metoprolol, timolol, nipradilol, penbutolol,nadolol, tilisolol, carvedilol, bisoprolol, betaxolol, celiprolol,bopindolol, bevantolol, labetalol, alprenolol, amosulalol, arotinolol,befunolol, bucumolol, bufetolol, buferalol, buprandolol, butylidine,butofilolol, carazolol, cetamolol, cloranolol, dilevalol, epanolol,levobunolol, mepindolol, metipranolol, moprolol, nadoxolol, nevibolol,oxprenolol, practol, pronetalol, sotalol, sufinalol, talindolol,tertalol, toliprolol, andxybenolol), calcium channel blocking drugs(such as aranidipine, efonidipine, nicardipine, bamidipine, benidipine,manidipine, cilnidipine, nisoldipine, nitrendipine, nifedipine,nilvadipine, felodipine, amlodipine, diltiazem, bepridil, clentiazem,phendiline, galopamil, mibefradil, prenylamine, semotiadil, terodiline,verapamil, cilnidipine, elgodipine, isradipine, lacidipine,lercanidipine, nimodipine, cinnarizine, flunarizine, lidoflazine,lomerizine, bencyclane, etafenone, and perhexyline) trimetazidine,dipyridamole, etafenone, dilazep, trapidil, nicorandil, enoxaparin, andaspirin. Examples of the diuretic include thiazide diuretics (such ashydrochlorothiazide, methyclothiazide, trichloromethiazide,benzylhydrochlorothiazide, and penflutizide), loop diuretics (such asfurosemide, etacrynic acid, bumetanide, piretanide, azosemide, andtorasemide), K⁺ sparing diuretics (spironolactone, triamterene,andpotassiumcanrenoate), osmotic diuretics (such as isosorbide,D-mannitol, and glycerin), nonthiazide diuretics (such as meticrane,tripamide, chlorthalidone, and mefruside), and acetazolamide. Examplesof the cardiotonic include digitalis formulations (such as digitoxin,digoxin, methyldigoxin, deslanoside, vesnarinone, lanatoside C, andproscillaridin), xanthine formulations (such as aminophylline, cholinetheophylline, diprophylline, and proxyphylline), catecholamineformulations (such as dopamine, dobutamine, and docarpamine), PDE IIIinhibitors (such as amrinone, olprinone, and milrinone), denopamine,ubidecarenone, pimobendan, levosimendan, aminoethylsulfonic acid,vesnarinone, carperitide, and colforsin daropate. Examples of theantiarrhythmic drug include ajmaline, pirmenol, procainamide,cibenzoline, disopyramide, quinidine, aprindine, mexiletine, lidocaine,phenyloin, pilsicainide, propafenone, flecainide, atenolol, acebutolol,sotalol, propranolol, metoprolol, pindolol, amiodarone, nifekalant,diltiazem, bepridil, and verapamil. Examples of the antihyperlipidemicdrug include atorvastatin, simvastatin, pravastatin sodium, fluvastatinsodium, clinofibrate, clofibrate, simfibrate, fenofibrate, bezafibrate,colestimide, and colestyramine. Examples of the immunosuppressantinclude azathioprine, mizoribine, cyclosporine, tacrolimus, gusperimus,and methotrexate.

Cell Death/Cancer

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to subjects who have recentlyreceived or are likely to receive a dose of radiation or toxin. In oneembodiment, the dose of radiation or toxin is received as part of awork-related or medical procedure, e.g., working in a nuclear powerplant, flying an airplane, an X-ray, CAT scan, or the administration ofa radioactive dye for medical imaging; in such an embodiment, thecompound is administered as a prophylactic measure. In anotherembodiment, the radiation or toxin exposure is received unintentionally,e.g., as a result of an industrial accident, habitation in a location ofnatural radiation, terrorist act, or act of war involving radioactive ortoxic material. In such a case, the compound is preferably administeredas soon as possible after the exposure to inhibit apoptosis and thesubsequent development of acute radiation syndrome.

Sirtuin-modulating compounds may also be used for treating and/orpreventing cancer. In certain embodiments, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedfor treating and/or preventing cancer. Calorie restriction has beenlinked to a reduction in the incidence of age-related disordersincluding cancer (see e.g., Bordone and Guarente, Nat. Rev. Mol. Cell.Biol. (2005 epub); Guarente and Picard, Cell 120: 473-82 (2005);Berrigan, et al., Carcinogenesis 23: 817-822 (2002); and Heilbronn andRavussin, Am. J. Clin. Nutr. 78: 361-369 (2003)). Additionally, the Sir2protein from yeast has been shown to be required for lifespan extensionby glucose restriction (see e.g., Lin et al., Science 289: 2126-2128(2000); Anderson et al., Nature 423: 181-185 (2003)), a yeast model forcalorie restriction. Accordingly, an increase in the level and/oractivity of a sirtuin protein may be useful for treating and/orpreventing the incidence of age-related disorders, such as, for example,cancer. In other embodiments, sirtuin-modulating compounds that decreasethe level and/or activity of a sirtuin protein may be used for treatingor preventing cancer. For example, inhibitory compounds may be used tostimulate acetylation of substrates such as p53 and thereby increaseapoptosis, as well as to reduce the lifespan of cells and organisms,render them more sensitive to stress, and/or increase theradiosensitivity and/or chemosensitivity of a cell or organism. Thus,inhibitory compounds may be used, e.g., for treating cancer. Exemplarycancers that may be treated using a sirtuin-modulating compound arethose of the brain and kidney; hormone-dependent cancers includingbreast, prostate, testicular, and ovarian cancers; lymphomas, andleukemias. In cancers associated with solid tumors, a modulatingcompound may be administered directly into the tumor. Cancer of bloodcells, e.g., leukemia, can be treated by administering a modulatingcompound into the blood stream or into the bone marrow. Benign cellgrowth can also be treated, e.g., warts. Other diseases that can betreated include autoimmune diseases, e.g., systemic lupus erythematosus,scleroderma, and arthritis, in which autoimmune cells should be removed.Viral infections such as herpes, HIV, adenovirus, and HTLV-1 associatedmalignant and benign disorders can also be treated by administration ofsirtuin-modulating compound. Alternatively, cells can be obtained from asubject, treated ex vivo to remove certain undesirable cells, e.g.,cancer cells, and administered back to the same or a different subject.

Chemotherapeutic agents that may be coadministered with modulatingcompounds described herein as having anti-cancer activity (e.g.,compounds that induce apoptosis, compounds that reduce lifespan orcompounds that render cells sensitive to stress) include:aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine,dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol,docetaxel, doxorabicin, epirubicin, estradiol, estramustine, etoposide,exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil,fluoxymesterone, flutamide, gemcitabine, genistein, goserelin,hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan,ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine,mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone,nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide,teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride,topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine,and vinorelbine.

These chemotherapeutic agents may be categorized by their mechanism ofaction into, for example, following groups: anti-metabolites/anti-canceragents, such as pyrimidine analogs (5-fluorouracil, floxuridine,capecitabine, gemcitabine and cytarabine) and purine analogs, folateantagonists and related inhibitors (mercaptopurine, thioguanine,pentostatin and 2-chlorodeoxyadenosine (cladribine));antiproliferative/antimitotic agents including natural products such asvinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubuledisruptors such as taxane (paclitaxel, docetaxel), vincristin,vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins(teniposide), DNA damaging agents (actinomycin, amsacrine,anthracyclines, bleomycin, busulfan, camptothecin, carboplatin,chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin,daunorubicin, docetaxel, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchloretlamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel,plicamycin, procarbazine, teniposide, triethylenethiophosphoramide andetoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, COX-2 inhibitors, dipyridamole,ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretoryagents (breveldin); immunosuppressives (cyclosporine, tacrolimus(FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (vascular endothelial growth factor (VEGF) inhibitors,fibroblast growth factor (FGF) inhibitors, epidermal growth factor (EGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan,irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; chromatin disruptors.

These chemotherapeutic agents may be used by themselves with asirtuin-modulating compound described herein as inducing cell death orreducing lifespan or increasing sensitivity to stress and/or incombination with other chemotherapeutics agents. Many combinatorialtherapies have been developed, including but not limited to those listedin Table 1.

TABLE 1 Exemplary combinatorial therapies for the treatment of cancer.Name Therapeutic agents ABV Doxorubicin, Bleomycin, Vinblastine ABVDDoxorubicin, Bleomycin, Vinblastine, Dacarbazine AC (Breast)Doxorubicin, Cyclophosphamide AC (Sarcoma) Doxorubicin, Cisplatin AC(Neuroblastoma) Cyclophosphamide, Doxorubicin ACE Cyclophosphamide,Doxorubicin, Etoposide ACe Cyclophosphamide, Doxorubicin AD Doxorubicin,Dacarbazine AP Doxorubicin, Cisplatin ARAC-DNR Cytarabine, DaunorubicinB-CAVe Bleomycin, Lomustine, Doxorubicin, Vinblastine BCVPP Carmustine,Cyclophosphamide, Vinblastine, Procarbazine, Prednisone BEACOPPBleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine,Procarbazine, Prednisone, Filgrastim BEP Bleomycin, Etoposide, CisplatinBIP Bleomycin, Cisplatin, Ifosfamide, Mesna BOMP Bleomycin, Vincristine,Cisplatin, Mitomycin CA Cytarabine, Asparaginase CABO Cisplatin,Methotrexate, Bleomycin, Vincristine CAF Cyclophosphamide, Doxorubicin,Fluorouracil CAL-G Cyclophosphamide, Daunorubicin, Vincristine,Prednisone, Asparaginase CAMP Cyclophosphamide, Doxorubicin,Methotrexate, Procarbazine CAP Cyclophosphamide, Doxorubicin, CisplatinCaT Carboplatin, Paclitaxel CAV Cyclophosphamide, Doxorubicin,Vincristine CAVE ADD CAV and Etoposide CA-VP16 Cyclophosphamide,Doxorubicin, Etoposide CC Cyclophosphamide, Carboplatin CDDP/VP-16Cisplatin, Etoposide CEF Cyclophosphamide, Epirubicin, FluorouracilCEPP(B) Cyclophosphamide, Etoposide, Prednisone, with or without/Bleomycin CEV Cyclophosphamide, Etoposide, Vincristine CF Cisplatin,Fluorouracil or Carboplatin Fluorouracil CHAP Cyclophosphamide orCyclophosphamide, Altretamine, Doxorubicin, Cisplatin ChlVPPChlorambucil, Vinblastine, Procarbazine, Prednisone CHOPCyclophosphamide, Doxorubicin, Vincristine, Prednisone CHOP-BLEO AddBleomycin to CHOP CISCA Cyclophosphamide, Doxorubicin, CisplatinCLD-BOMP Bleomycin, Cisplatin, Vincristine, Mitomycin CMF Methotrexate,Fluorouracil, Cyclophosphamide CMFP Cyclophosphamide, Methotrexate,Fluorouracil, Prednisone CMFVP Cyclophosphamide, Methotrexate,Fluorouracil, Vincristine, Prednisone CMV Cisplatin, Methotrexate,Vinblastine CNF Cyclophosphamide, Mitoxantrone, Fluorouracil CNOPCyclophosphamide, Mitoxantrone, Vincristine, Prednisone COB Cisplatin,Vincristine, Bleomycin CODE Cisplatin, Vincristine, Doxorubicin,Etoposide COMLA Cyclophosphamide, Vincristine, Methotrexate, Leucovorin,Cytarabine COMP Cyclophosphamide, Vincristine, Methotrexate, PrednisoneCooper Regimen Cyclophosphamide, Methotrexate, Fluorouracil,Vincristine, Prednisone COP Cyclophosphamide, Vincristine, PrednisoneCOPE Cyclophosphamide, Vincristine, Cisplatin, Etoposide COPPCyclophosphamide, Vincristine, Procarbazine, Prednisone CP(Chroniclymphocytic Chlorambucil, Prednisone leukemia) CP (Ovarian Cancer)Cyclophosphamide, Cisplatin CT Cisplatin, Paclitaxel CVD Cisplatin,Vinblastine, Dacarbazine CVI Carboplatin, Etoposide, Ifosfamide, MesnaCVP Cyclophosphamide, Vincristine, Prednisome CVPP Lomustine,Procarbazine, Prednisone CYVADIC Cyclophosphamide, Vincristine,Doxorubicin, Dacarbazine DA Daunorubicin, Cytarabine DAT Daunorubicin,Cytarabine, Thioguanine DAV Daunorubicin, Cytarabine, Etoposide DCTDaunorubicin, Cytarabine, Thioguanine DHAP Cisplatin, Cytarabine,Dexamethasone DI Doxorubicin, Ifosfamide DTIC/Tamoxifen Dacarbazine,Tamoxifen DVP Daunorubicin, Vincristine, Prednisone EAP Etoposide,Doxorubicin, Cisplatin EC Etoposide, Carboplatin EFP Etoposie,Fluorouracil, Cisplatin ELF Etoposide, Leucovorin, Fluorouracil EMA 86Mitoxantrone, Etoposide, Cytarabine EP Etoposide, Cisplatin EVAEtoposide, Vinblastine FAC Fluorouracil, Doxorubicin, CyclophosphamideFAM Fluorouracil, Doxorubicin, Mitomycin FAMTX Methotrexate, Leucovorin,Doxorubicin FAP Fluorouracil, Doxorubicin, Cisplatin F-CL Fluorouracil,Leucovorin FEC Fluorouracil, Cyclophosphamide, Epirubicin FEDFluorouracil, Etoposide, Cisplatin FL Flutamide, Leuprolide FZFlutamide, Goserelin acetate implant HDMTX Methotrexate, LeucovorinHexa-CAF Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil ICE-TIfosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna IDMTX/6-MPMethotrexate, Mercaptopurine, Leucovorin IE Ifosfamide, Etoposie, MesnaIfoVP Ifosfamide, Etoposide, Mesna IPA Ifosfamide, Cisplatin,Doxorubicin M-2 Vincristine, Carmustine, Cyclophosphamide, Prednisone,Melphalan MAC-III Methotrexate, Leucovorin, Dactinomycin,Cyclophosphamide MACC Methotrexate, Doxorubicin, Cyclophosphamide,Lomustine MACOP-B Methotrexate, Leucovorin, Doxorubicin,Cyclophosphamide, Vincristine, Bleomycin, Prednisone MAID Mesna,Doxorubicin, Ifosfamide, Dacarbazine m-BACOD Bleomycin, Doxorubicin,Cyclophosphamide, Vincristine, Dexamethasone, Methotrexate, LeucovorinMBC Methotrexate, Bleomycin, Cisplatin MC Mitoxantrone, Cytarabine MFMethotrexate, Fluorouracil, Leucovorin MICE Ifosfamide, Carboplatin,Etoposide, Mesna MINE Mesna, Ifosfamide, Mitoxantrone, Etoposidemini-BEAM Carmustine, Etoposide, Cytarabine, Melphalan MOBP Bleomycin,Vincristine, Cisplatin, Mitomycin MOP Mechlorethamine, Vincristine,Procarbazine MOPP Mechlorethamine, Vincristine, Procarbazine, PrednisoneMOPP/ABV Mechlorethamine, Vincristine, Procarbazine, Prednisone,Doxorubicin, Bleomycin, Vinblastine MP (multiple myeloma) Melphalan,Prednisone MP (prostate cancer) Mitoxantrone, Prednisone MTX/6-MOMethotrexate, Mercaptopurine MTX/6-MP/VP Methotrexate, Mercaptopurine,Vincristine, Prednisone MTX-CDDPAdr Methotrexate, Leucovorin, Cisplatin,Doxorubicin MV (breast cancer) Mitomycin, Vinblastine MV (acutemyelocytic Mitoxantrone, Etoposide leukemia) M-VAC MethotrexateVinblastine, Doxorubicin, Cisplatin MVP Mitomycin Vinblastine, CisplatinMVPP Mechlorethamine, Vinblastine, Procarbazine, Prednisone NFLMitoxantrone, Fluorouracil, Leucovorin NOVP Mitoxantrone, Vinblastine,Vincristine OPA Vincristine, Prednisone, Doxorubicin OPPA AddProcarbazine to OPA. PAC Cisplatin, Doxorubicin PAC-I Cisplatin,Doxorubicin, Cyclophosphamide PA-CI Cisplatin, Doxorubicin PCPaclitaxel, Carboplatin or Paclitaxel, Cisplatin PCV Lomustine,Procarbazine, Vincristine PE Paclitaxel, Estramustine PFL Cisplatin,Fluorouracil, Leucovorin POC Prednisone, Vincristine, Lomustine ProMACEPrednisone, Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide,Etoposide ProMACE/cytaBOM Prednisone, Doxorubicin, Cyclophosphamide,Etoposide, Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin,Cotrimoxazole PRoMACE/MOPP Prednisone, Doxorubicin, Cyclophosphamide,Etoposide, Mechlorethamine, Vincristine, Procarbazine, Methotrexate,Leucovorin Pt/VM Cisplatin, Teniposide PVA Prednisone, Vincristine,Asparaginase PVB Cisplatin, Vinblastine, Bleomycin PVDA Prednisone,Vincristine, Daunorubicin, Asparaginase SMF Streptozocin, Mitomycin,Fluorouracil TAD Mechlorethamine, Doxorubicin, Vinblastine, Vincristine,Bleomycin, Etoposide, Prednisone TCF Paclitaxel, Cisplatin, FluorouracilTIP Paclitaxel, Ifosfamide, Mesna, Cisplatin TTT Methotrexate,Cytarabine, Hydrocortisone Topo/CTX Cyclophosphamide, Topotecan, MesnaVAB-6 Cyclophosphamide, Dactinomycin, Vinblastine, Cisplatin, BleomycinVAC Vincristine, Dactinomycin, Cyclophosphamide VACAdr Vincristine,Cyclophosphamide, Doxorubicin, Dactinomycin, Vincristine VADVincristine, Doxorubicin, Dexamethasone VATH Vinblastine, Doxorubicin,Thiotepa, Flouxymesterone VBAP Vincristine, Carmustine, Doxorubicin,Prednisone VBCMP Vincristine, Carmustine, Melphalan, Cyclophosphamide,Prednisone VC Vinorelbine, Cisplatin VCAP Vincristine, Cyclophosphamide,Doxorubicin, Prednisone VD Vinorelbine, Doxorubicin VelP Vinblastine,Cisplatin, Ifosfamide, Mesna VIP Etoposide, Cisplatin, Ifosfamide, MesnaVM Mitomycin, Vinblastine VMCP Vincristine, Melphalan, Cyclophosphamide,Prednisone VP Etoposide, Cisplatin V-TAD Etoposide, Thioguanine,Daunorubicin, Cytarabine 5 + 2 Cytarabine, Daunorubicin, Mitoxantrone7 + 3 Cytarabine with/, Daunorubicin or Idarubicin or Mitoxantrone “8 in1” Methylprednisolone, Vincristine, Lomustine, Procarbazine,Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine

In addition to conventional chemotherapeutics, the sirtuin-modulatingcompounds described herein as capable of inducing cell death or reducinglifespan can also be used with antisense RNA, RNAi or otherpolynucleotides to inhibit the expression of the cellular componentsthat contribute to unwanted cellular proliferation that are targets ofconventional chemotherapy. Such targets are, merely to illustrate,growth factors, growth factor receptors, cell cycle regulatory proteins,transcription factors, or signal transduction kinases.

Combination therapies comprising sirtuin-modulating compounds and aconventional chemotherapeutic agent may be advantageous over combinationtherapies known in the art because the combination allows theconventional chemotherapeutic agent to exert greater effect at lowerdosage. In a preferred embodiment, the effective dose (ED₅₀) for achemotherapeutic agent, or combination of conventional chemotherapeuticagents, when used in combination with a sirtuin-modulating compound isat least 2 fold less than the ED₅₀ for the chemotherapeutic agent alone,and even more preferably at 5 fold, 10 fold or even 25 fold less.Conversely, the therapeutic index (TI) for such chemotherapeutic agentor combination of such chemotherapeutic agent when used in combinationwith a sirtuin-modulating compound described herein can be at least 2fold greater than the TI for conventional chemotherapeutic regimenalone, and even more preferably at 5 fold, 10 fold or even 25 foldgreater.

Neuronal Diseases/Disorders

In certain aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat patientssuffering from neurodegenerative diseases, and traumatic or mechanicalinjury to the central nervous system (CNS), spinal cord or peripheralnervous system (PNS). Neurodegenerative disease typically involvesreductions in the mass and volume of the human brain, which may be dueto the atrophy and/or death of brain cells, which are far more profoundthan those in a healthy person that are attributable to aging.Neurodegenerative diseases can evolve gradually, after a long period ofnormal brain function, due to progressive degeneration (e.g., nerve celldysfunction and death) of specific brain regions. Alternatively,neurodegenerative diseases can have a quick onset, such as thoseassociated with trauma or toxins. The actual onset of brain degenerationmay precede clinical expression by many years. Examples ofneurodegenerative diseases include, but are not limited to, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewybody disease, chorea-acanthocytosis, primary lateral sclerosis, oculardiseases (ocular neuritis), chemotherapy-induced neuropathies (e.g.,from vincristine, paclitaxel, bortezomib), diabetes-induced neuropathiesand Friedreich's ataxia. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can be used to treat thesedisorders and others as described below.

AD is a chronic, incurable, and unstoppable CNS disorder that occursgradually, resulting in memory loss, unusual behavior, personalitychanges, and a decline in thinking abilities. These losses are relatedto the death of specific types of brain cells and the breakdown ofconnections and their supporting network (e.g. glial cells) betweenthem. AD has been described as childhood development in reverse. In mostpeople with AD, symptoms appear after the age 60. The earliest symptomsinclude loss of recent memory, faulty judgment, and changes inpersonality. Later in the disease, those with AD may forget how to dosimple tasks like washing their hands. Eventually people with AD loseall reasoning abilities and become dependent on other people for theireveryday care. Finally, the disease becomes so debilitating thatpatients are bedridden and typically develop coexisting illnesses.

PD is a chronic, incurable, and unstoppable CNS disorder that occursgradually and results in uncontrolled body movements, rigidity, tremor,and dyskinesia. These motor system problems are related to the death ofbrain cells in an area of the brain that produces dopamine, a chemicalthat helps control muscle activity. In most people with PD, symptomsappear after age 50. The initial symptoms of PD are a pronounced tremoraffecting the extremities, notably in the hands or lips. Subsequentcharacteristic symptoms of PD are stiffness or slowness of movement, ashuffling walk, stooped posture, and impaired balance. There are wideranging secondary symptoms such as memory loss, dementia, depression,emotional changes, swallowing difficulties, abnormal speech, sexualdysfunction, and bladder and bowel problems. These symptoms will beginto interfere with routine activities, such as holding a fork or readinga newspaper. Finally, people with PD become so profoundly disabled thatthey are bedridden.

ALS (motor neuron disease) is a chronic, incurable, and unstoppable CNSdisorder that attacks the motor neurons, components of the CNS thatconnect the brain to the skeletal muscles. In ALS, the motor neuronsdeteriorate and eventually die, and though a person's brain normallyremains fully functioning and alert, the command to move never reachesthe muscles. Most people who get ALS are between 40 and 70 years old.The first motor neurons that weaken are those controlling the arms orlegs. Those with ALS may have trouble walking, they may drop things,fall, slur their speech, and laugh or cry uncontrollably. Eventually themuscles in the limbs begin to atrophy from disuse. This muscle weaknesswill become debilitating and a person will need a wheel chair or becomeunable to function out of bed.

The causes of these neurological diseases have remained largely unknown.They are conventionally defined as distinct diseases, yet clearly showextraordinary similarities in basic processes and commonly demonstrateoverlapping symptoms far greater than would be expected by chance alone.Current disease definitions fail to properly deal with the issue ofoverlap and a new classification of the neurodegenerative disorders hasbeen called for.

HD is another neurodegenerative disease resulting from geneticallyprogrammed degeneration of neurons in certain areas of the brain. Thisdegeneration causes uncontrolled movements, loss of intellectualfaculties, and emotional disturbance. HD is a familial disease, passedfrom parent to child through a dominant mutation in the wild-type gene.Some early symptoms of HD are mood swings, depression, irritability ortrouble driving, learning new things, remembering a fact, or making adecision. As the disease progresses, concentration on intellectual tasksbecomes increasingly difficult and the patient may have difficultyfeeding himself or herself and swallowing.

Tay-Sachs disease and Sandhoff disease are glycolipid storage diseasescaused by the lack of lysosomal β-hexosaminidase (Gravel et al., in TheMetabolic Basis of Inherited Disease, eds. Scriver et al., McGraw-Hill,New York, pp. 2839-2879, 1995). In both disorders, GM2 ganglioside andrelated glycolipidssubstrates for hexosaminidase accumulate in thenervous system and trigger acute neurodegeneration. In the most severeforms, the onset of symptoms begins in early infancy. A precipitousneurodegenerative course then ensues, with affected infants exhibitingmotor dysfunction, seizure, visual loss, and deafness. Death usuallyoccurs by 2-5 years of age. Neuronal loss through an apoptotic mechanismhas been demonstrated (Huang et al., Hum. Mol. Genet. 6: 1879-1885,1997).

It is well-known that apoptosis plays a role in AIDS pathogenesis in theimmune system. However, HIV-1 also induces neurological disease. Shi etal. (J. Clin. Invest. 98: 1979-1990, 1996) examined apoptosis induced byHIV-1 infection of the CNS in an in vitro model and in brain tissue fromAIDS patients, and found that HIV-1 infection of primary brain culturesinduced apoptosis in neurons and astrocytes in vitro. Apoptosis ofneurons and astrocytes was also detected in brain tissue from 10/11 AIDSpatients, including 5/5 patients with HIV-1 dementia and 4/5 nondementedpatients.

There are four main peripheral neuropathies associated with HIV, namelysensory neuropathy, AIDP/CIPD, drug-induced neuropathy and CMV-related.

The most common type of neuropathy associated with AIDS is distalsymmetrical polyneuropathy (DSPN). This syndrome is a result of nervedegeneration and is characterized by numbness and a sensation of pinsand needles. DSPN causes few serious abnormalities and mostly results innumbness or tingling of the feet and slowed reflexes at the ankles. Itgenerally occurs with more severe immunosuppression and is steadilyprogressive. Treatment with tricyclic antidepressants relieves symptomsbut does not affect the underlying nerve damage.

A less frequent, but more severe type of neuropathy is known as acute orchronic inflammatory demyelinating polyneuropathy (AIDP/CIDP). InAIDP/CIDP there is damage to the fatty membrane covering the nerveimpulses. This kind of neuropathy involves inflammation and resemblesthe muscle deterioration often identified with long-term use of AZT. Itcan be the first manifestation of HIV infection, where the patient maynot complain of pain, but fails to respond to standard reflex tests.This kind of neuropathy may be associated with seroconversion, in whichcase it can sometimes resolve spontaneously. It can serve as a sign ofHIV infection and indicate that it might be time to consider antiviraltherapy. AIDP/CIDP may be auto-immune in origin.

Drug-induced, or toxic, neuropathies can be very painful. Antiviraldrugs commonly cause peripheral neuropathy, as do other drugs e.g.vincristine, dilantin (an anti-seizure medication), high-dose vitamins,isoniazid, and folic acid antagonists. Peripheral neuropathy is oftenused in clinical trials for antivirals as a dose-limiting side effect,which means that more drugs should not be administered. Additionally,the use of such drugs can exacerbate otherwise minor neuropathies.Usually, these drug-induced neuropathies are reversible with thediscontinuation of the drug.

CMV causes several neurological syndromes in AIDS, includingencephalitis, myelitis, and polyradiculopathy.

Neuronal loss is also a salient feature of prion diseases, such asCreutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease),Scrapie Disease in sheep and goats, and feline spongiform encephalopathy(FSE) in cats. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for treating orpreventing neuronal loss due to these prior diseases.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used to treat orprevent any disease or disorder involving axonopathy. Distal axonopathyis a type of peripheral neuropathy that results from some metabolic ortoxic derangement of peripheral nervous system (PNS) neurons. It is themost common response of nerves to metabolic or toxic disturbances, andas such may be caused by metabolic diseases such as diabetes, renalfailure, deficiency syndromes such as malnutrition and alcoholism, orthe effects of toxins or drugs. The most common cause of distalaxonopathy is diabetes, and the most common distal axonopathy isdiabetic neuropathy. The most distal portions of axons are usually thefirst to degenerate, and axonal atrophy advances slowly towards thenerve's cell body. If the noxious stimulus is removed, regeneration ispossible, though prognosis decreases depending on the duration andseverity of the stimulus. Those with distal axonopathies usually presentwith symmetrical glove-stocking sensori-motor disturbances. Deep tendonreflexes and autonomic nervous system (ANS) functions are also lost ordiminished in affected areas.

Diabetic neuropathies are neuropathic disorders that are associated withdiabetes mellitus. These conditions usually result from diabeticmicrovascular injury involving small blood vessels that supply nerves(vasa nervorum). Relatively common conditions which may be associatedwith diabetic neuropathy include third nerve palsy; mononeuropathy;mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy;autonomic neuropathy; and thoracoabdominal neuropathy. Clinicalmanifestations of diabetic neuropathy include, for example, sensorimotorpolyneuropathy such as numbness, sensory loss, dysesthesia and nighttimepain; autonomic neuropathy such as delayed gastric emptying orgastroparesis; and cranial neuropathy such as oculomotor (3rd)neuropathies or Mononeuropathies of the thoracic or lumbar spinalnerves.

Peripheral neuropathy is the medical term for damage to nerves of theperipheral nervous system, which may be caused either by diseases of thenerve or from the side-effects of systemic illness. Peripheralneuropathies vary in their presentation and origin, and may affect thenerve or the neuromuscular junction. Major causes of peripheralneuropathy include seizures, nutritional deficiencies, and HIV, thoughdiabetes is the most likely cause. Mechanical pressure from staying inone position for too long, a tumor, intraneural hemorrhage, exposing thebody to extreme conditions such as radiation, cold temperatures, ortoxic substances can also cause peripheral neuropathy.

In an exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treat orprevent multiple sclerosis (MS), including relapsing MS andmonosymptomatic MS, and other demyelinating conditions, such as, forexample, chromic inflammatory demyelinating polyneuropathy (CIDP), orsymptoms associated therewith.

MS is a chronic, often disabling disease of the central nervous system.Various and converging lines of evidence point to the possibility thatthe disease is caused by a disturbance in the immune function, althoughthe cause of this disturbance has not been established. This disturbancepermits cells of the immune system to “attack” myelin, the fatcontaining insulating sheath that surrounds the nerve axons located inthe central nervous system (“CNS”). When myelin is damaged, electricalpulses cannot travel quickly or normally along nerve fiber pathways inthe brain and spinal cord. This results in disruption of normalelectrical conductivity within the axons, fatigue and disturbances ofvision, strength, coordination, balance, sensation, and bladder andbowel function.

As such, MS is now a common and well-known neurological disorder that ischaracterized by episodic patches of inflammation and demyelinationwhich can occur anywhere in the CNS. However, almost always without anyinvolvement of the peripheral nerves associated therewith. Demyelinationproduces a situation analogous to that resulting from cracks or tears inan insulator surrounding an electrical cord. That is, when theinsulating sheath is disrupted, the circuit is “short circuited” and theelectrical apparatus associated therewith will function intermittentlyor nor at all. Such loss of myelin surrounding nerve fibers results inshort circuits in nerves traversing the brain and the spinal cord thatthereby result in symptoms of MS. It is further found that suchdemyelination occurs in patches, as opposed to along the entire CNS. Inaddition, such demyelination may be intermittent. Therefore, suchplaques are disseminated in both time and space.

It is believed that the pathogenesis involves a local disruption of theblood brain barrier which causes a localized immune and inflammatoryresponse, with consequent damage to myelin and hence to neurons.

Clinically, MS exists in both sexes and can occur at any age. However,its most common presentation is in the relatively young adult, oftenwith a single focal lesion such as a damage of the optic nerve, an areaof anesthesia (loss of sensation), or paraesthesia (localize loss offeeling), or muscular weakness. In addition, vertigo, double vision,localized pain, incontinence, and pain in the arms and legs may occurupon flexing of the neck, as well as a large variety of less commonsymptoms.

An initial attack of MS is often transient, and it may be weeks, months,or years before a further attack occurs. Some individuals may enjoy astable, relatively event free condition for a great number of years,while other less fortunate ones may experience a continual downhillcourse ending in complete paralysis. There is, most commonly, a seriesof remission and relapses, in which each relapse leaves a patientsomewhat worse than before. Relapses may be triggered by stressfulevents, viral infections or toxins. Therein, elevated body temperature,i.e., a fever, will make the condition worse, or as a reduction oftemperature by, for example, a cold bath, may make the condition better.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treattrauma to the nerves, including, trauma due to disease, injury(including surgical intervention), or environmental trauma (e.g.,neurotoxins, alcoholism, etc.).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be useful to prevent, treat, and alleviatesymptoms of various PNS disorders, such as the ones described below. ThePNS is composed of the nerves that lead to or branch off from the spinalcord and CNS. The peripheral nerves handle a diverse array of functionsin the body, including sensory, motor, and autonomic functions. When anindividual has a peripheral neuropathy, nerves of the PNS have beendamaged. Nerve damage can arise from a number of causes, such asdisease, physical injury, poisoning, or malnutrition. These agents mayaffect either afferent or efferent nerves. Depending on the cause ofdamage, the nerve cell axon, its protective myelin sheath, or both maybe injured or destroyed.

The term “peripheral neuropathy” encompasses a wide range of disordersin which the nerves outside of the brain and spinal cord—peripheralnerves—have been damaged. Peripheral neuropathy may also be referred toas peripheral neuritis, or if many nerves are involved, the termspolyneuropathy or polyneuritis may be used.

Peripheral neuropathy is a widespread disorder, and there are manyunderlying causes. Some of these causes are common, such as diabetes,and others are extremely rare, such as acrylamide poisoning and certaininherited disorders. The most common worldwide cause of peripheralneuropathy is leprosy. Leprosy is caused by the bacterium Mycobacteriumleprae, which attacks the peripheral nerves of affected people.

Leprosy is extremely rare in the United States, where diabetes is themost commonly known cause of peripheral neuropathy. It has beenestimated that more than 17 million people in the United States andEurope have diabetes-related polyneuropathy. Many neuropathies areidiopathic; no known cause can be found. The most common of theinherited peripheral neuropathies in the United States isCharcot-Marie-Tooth disease, which affects approximately 125,000persons.

Another of the better known peripheral neuropathies isGuillain-Barrésyndrome, which arises from complications associated withviral illnesses, such as cytomegalovirus, Epstein-Barr virus, and humanimmunodeficiency virus (HIV), or bacterial infection, includingCampylobacter jejuni and Lyme disease. The worldwide incidence rate isapproximately 1.7 cases per 100,000 people annually. Other well-knowncauses of peripheral neuropathies include chronic alcoholism, infectionof the varicella-zoster virus, botulism, and poliomyelitis. Peripheralneuropathy may develop as a primary symptom, or it may be due to anotherdisease. For example, peripheral neuropathy is only one symptom ofdiseases such as amyloid neuropathy, certain cancers, or inheritedneurologic disorders. Such diseases may affect the PNS and the CNS, aswell as other body tissues.

Other PNS diseases treatable with sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein include:Brachial Plexus Neuropathies (diseases of the cervical and firstthoracic roots, nerve trunks, cords, and peripheral nerve components ofthe brachial plexus. Clinical manifestations include regional pain,paresthesia; muscle weakness, and decreased sensation in the upperextremity. These disorders may be associated with trauma, includingbirth injuries; thoracic outlet syndrome; neoplasms, neuritis,radiotherapy; and other conditions. See Adams et al., Principles ofNeurology, 6th ed, pp 1351-2); Diabetic Neuropathies (peripheral,autonomic, and cranial nerve disorders that are associated with diabetesmellitus). These conditions usually result from diabetic microvascularinjury involving small blood vessels that supply nerves (vasa nervorum).Relatively common conditions which may be associated with diabeticneuropathy include third nerve palsy; mononeuropathy; mononeuritismultiplex; diabetic amyotrophy; a painful polyneuropathy; autonomicneuropathy; and thoracoabdominal neuropathy (see Adams et al.,Principles of Neurology, 6th ed, p 1325); mononeuropathies (disease ortrauma involving a single peripheral nerve in isolation, or out ofproportion to evidence of diffuse peripheral nerve dysfunction).Mononeuritis multiplex refers to a condition characterized by multipleisolated nerve injuries. Mononeuropathies may result from a wide varietyof causes, including ischemia; traumatic injury; compression; connectivetissue diseases; cumulative trauma disorders; and other conditions;Neuralgia (intense or aching pain that occurs along the course ordistribution of a peripheral or cranial nerve); Peripheral NervousSystem Neoplasms (neoplasms which arise from peripheral nerve tissue).This includes neurofibromas; Schwannomas; granular cell tumors; andmalignant peripheral nerve sheath tumors (see DeVita Jr et al., Cancer:Principles and Practice of Oncology, 5th ed, pp 1750-1); and NerveCompression Syndromes (mechanical compression of nerves or nerve rootsfrom internal or external causes. These may result in a conduction blockto nerve impulses, due to, for example, myelin sheath dysfunction, oraxonal loss. The nerve and nerve sheath injuries may be caused byischemia; inflammation; or a direct mechanical effect; Neuritis (ageneral term indicating inflammation of a peripheral or cranial nerve).Clinical manifestation may include pain; paresthesias; paresis; orhyperesthesia; Polyneuropathies (diseases of multiple peripheralnerves). The various forms are categorized by the type of nerve affected(e.g., sensory, motor, or autonomic), by the distribution of nerveinjury (e.g., distal vs. proximal), by nerve component primarilyaffected (e.g., demyelinating vs. axonal), by etiology, or by pattern ofinheritance.

In another embodiment, a sirtuin activating compound may be used totreat or prevent chemotherapeutic induced neuropathy. The sirtuinmodulating compounds may be administered prior to administration of thechemotherapeutic agent, concurrently with administration of thechemotherapeutic drug, and/or after initiation of administration of thechemotherapeutic drug. If the sirtuin activating compound isadministered after the initiation of administration of thechemotherapeutic drug, it is desirable that the sirtuin activatingcompound be administered prior to, or at the first signs, ofchemotherapeutic induced neuropathy.

Chemotherapy drugs can, damage any part of the nervous system.Encephalopathy and myelopathy are fortunately very rare. Damage toperipheral nerves is much more common and can be a side effect oftreatment experienced by people with cancers, such as lymphoma. Most ofthe neuropathy affects sensory rather than motor nerves. Thus, thecommon symptoms are tingling, numbness or a loss of balance. The longestnerves in the body seem to be most sensitive hence the fact that mostpatients will report numbness or pins and needles in their hands andfeet.

The chemotherapy drugs which are most commonly associated withneuropathy, are the Vinca alkaloids (anti-cancer drugs originallyderived from a member of the periwinkle—the Vinca plant genus) and aplatinum-containing drug called Cisplatin. The Vinca alkaloids includethe drugs vinblastine, vincristine and vindesine. Many combinationchemotherapy treatments for lymphoma for example CHOP and CVP containvincristine, which is the drug known to cause this problem mostfrequently. Indeed, it is the risk of neuropathy that limits the dose ofvincristine that can be administered.

Studies that have been performed have shown that most patients will losesome reflexes in their legs as a result of treatment with vincristineand many will experience some degree of tingling (paresthesia) in theirfingers and toes. The neuropathy does not usually manifest itself rightat the start of the treatment but generally comes on over a period of afew weeks. It is not essential to stop the drug at the first onset ofsymptoms, but if the neuropathy progresses this may be necessary. It isvery important that patients should report such symptoms to theirdoctors, as the nerve damage is largely reversible if the drug isdiscontinued. Most doctors will often reduce the dose of vincristine orswitch to another form of Vinca alkaloid such as vinblastine orvindesine if the symptoms are mild. Occasionally, the nerves supplyingthe bowel are affected causing abdominal pain and constipation.

In another embodiment, a sirtuin activating compound may be used totreat or prevent a polyglutamine disease. Huntington's Disease (HD) andSpinocerebellar ataxia type 1 (SCA1) are just two examples of a class ofgenetic diseases caused by dynamic mutations involving the expansion oftriplet sequence repeats. In reference to this common mechanism, thesedisorders are called trinucleotide repeat diseases. At least 14 suchdiseases are known to affect human beings. Nine of them, including SCA1and Huntington's disease, have CAG as the repeated sequence (see Table 2below). Since CAG codes for an amino acid called glutamine, these ninetrinucleotide repeat disorders are collectively known as polyglutaminediseases.

Although the genes involved in different polyglutamine diseases havelittle in common, the disorders they cause follow a strikingly similarcourse. Each disease is characterized by a progressive degeneration of adistinct group of nerve cells. The major symptoms of these diseases aresimilar, although not identical, and usually affect people in midlife.Given the similarities in symptoms, the polyglutamine diseases arehypothesized to progress via common cellular mechanisms. In recentyears, scientists have made great strides in unraveling what themechanisms are.

Above a certain threshold, the greater the number of glutamine repeatsin a protein, the earlier the onset of disease and the more severe thesymptoms. This suggests that abnormally long glutamine tracts rendertheir host protein toxic to nerve cells.

To test this hypothesis, scientists have generated geneticallyengineered mice expressing proteins with long polyglutamine tracts.Regardless of whether the mice express full-length proteins or onlythose portions of the proteins containing the polyglutamine tracts, theydevelop symptoms of polyglutamine diseases. This suggests that a longpolyglutamine tract by itself is damaging to cells and does not have tobe part of a functional protein to cause its damage.

For example, it is thought that the symptoms of SCA1 are not directlycaused by the loss of normal ataxin-1 function but rather by theinteraction between ataxin-1 and another protein called LANP. LANP isneeded for nerve cells to communicate with one another and thus fortheir survival. When the mutant ataxin-1 protein accumulates insidenerve cells, it “traps” the LANP protein, interfering with its normalfunction. After a while, the absence of LANP function appears to causenerve cells to malfunction.

TABLE 2 Summary of Polyglutamine Diseases. Normal Disease GeneChromosomal Pattern of repeat repeat Disease name location inheritanceProtein length length Spinobulbar AR Xq13-21 X-linked androgen 9-3638-62 muscular recessive receptor atrophy (AR) (Kennedy disease)Huntington's HD 4p16.3 autosomal huntingtin 6-35  36-121 diseasedominant Dentatorubral- DRPLA 12p13.31 autosomal atrophin-1 6-35 49-88pallidoluysian dominant atrophy (Haw River syndrome) SpinocerebellarSCA1 6p23 autosomal ataxin-1 6-44 39-82 ataxia type 1 dominantSpinocerebellar SCA2 12q24.1 autosomal ataxin-2 15-31  36-63 ataxia type2 dominant Spinocerebellar SCA3 14q32.1 autosomal ataxin-3 12-40  55-84ataxia type 3 dominant (Machado- Joseph disease) Spinocerebellar SCA619p13 autosomal α1_(A)- 4-18 21-33 ataxia type 6 dominant voltage-dependent calcium channel subunit Spinocerebellar SCA7 3p12-13 autosomalataxin-7 4-35  37-306 ataxia type 7 dominant Spinocerebellar SCA17 6q27autosomal TATA 25-42  45-63 ataxia type 17 dominant binding protein

Many transcription factors have also been found in neuronal inclusionsin different diseases. It is possible that these transcription factorsinteract with the polyglutamine-containing proteins and then becometrapped in the neuronal inclusions. This in turn might keep thetranscription factors from turning genes on and off as needed by thecell. Another observation is hypoacetylation of histones in affectedcells. This has led to the hypothesis that Class I/II HistoneDeacetylase (HDAC I/II) inhibitors, which are known to increase histoneacetylation, may be a novel therapy for polyglutamine diseases (U.S.patent application Ser. No. 10/476,627; “Method of treatingneurodegenerative, psychiatric, and other disorders with deacetylaseinhibitors”).

In yet another embodiment, the invention provides a method for treatingor preventing neuropathy related to ischemic injuries or diseases, suchas, for example, coronary heart disease (including congestive heartfailure and myocardial infarctions), stroke, emphysema, hemorrhagicshock, peripheral vascular disease (upper and lower extremities) andtransplant related injuries.

In certain embodiments, the invention provides a method to treat acentral nervous system cell to prevent damage in response to a decreasein blood flow to the cell. Typically the severity of damage that may beprevented will depend in large part on the degree of reduction in bloodflow to the cell and the duration of the reduction. By way of example,the normal amount of perfusion to brain gray matter in humans is about60 to 70 mL/100 g of brain tissue/min. Death of central nervous systemcells typically occurs when the flow of blood falls below approximately8-10 mL/100 g of brain tissue/min, while at slightly higher levels (i.e.20-35 mL/100 g of brain tissue/min) the tissue remains alive but notable to function. In one embodiment, apoptotic or necrotic cell deathmay be prevented. In still a further embodiment, ischemic-mediateddamage, such as cytoxic edema or central nervous system tissue anoxemia,may be prevented. In each embodiment, the central nervous system cellmay be a spinal cell or a brain cell.

Another aspect encompasses administrating a sirtuin activating compoundto a subject to treat a central nervous system ischemic condition. Anumber of central nervous system ischemic conditions may be treated bythe sirtuin activating compounds described herein. In one embodiment,the ischemic condition is a stroke that results in any type of ischemiccentral nervous system damage, such as apoptotic or necrotic cell death,cytoxic edema or central nervous system tissue anoxia. The stroke mayimpact any area of the brain or be caused by any etiology commonly knownto result in the occurrence of a stroke. In one alternative of thisembodiment, the stroke is a brain stem stroke. Generally speaking, brainstem strokes strike the brain stem, which control involuntarylife-support functions such as breathing, blood pressure, and heartbeat.In another alternative of this embodiment, the stroke is a cerebellarstroke. Typically, cerebellar strokes impact the cerebellum area of thebrain, which controls balance and coordination. In still anotherembodiment, the stroke is an embolic stroke. In general terms, embolicstrokes may impact any region of the brain and typically result from theblockage of an artery by a vaso-occlusion. In yet another alternative,the stroke may be a hemorrhagic stroke. Like ischemic strokes,hemorrhagic stroke may impact any region of the brain, and typicallyresult from a ruptured blood vessel characterized by a hemorrhage(bleeding) within or surrounding the brain. In a further embodiment, thestroke is a thrombotic stroke. Typically, thrombotic strokes result fromthe blockage of a blood vessel by accumulated deposits.

In another embodiment, the ischemic condition may result from a disorderthat occurs in a part of the subject's body outside of the centralnervous system, but yet still causes a reduction in blood flow to thecentral nervous system. These disorders may include, but are not limitedto a peripheral vascular disorder, a venous thrombosis, a pulmonaryembolus, arrhythmia (e.g. atrial fibrillation), a myocardial infarction,a transient ischemic attack, unstable angina, or sickle cell anemia.Moreover, the central nervous system ischemic condition may occur asresult of the subject undergoing a surgical procedure. By way ofexample, the subject may be undergoing heart surgery, lung surgery,spinal surgery, brain surgery, vascular surgery, abdominal surgery, ororgan transplantation surgery. The organ transplantation surgery mayinclude heart, lung, pancreas, kidney or liver transplantation surgery.Moreover, the central nervous system ischemic condition may occur as aresult of a trauma or injury to a part of the subject's body outside thecentral nervous system. By way of example, the trauma or injury maycause a degree of bleeding that significantly reduces the total volumeof blood in the subject's body. Because of this reduced total volume,the amount of blood flow to the central nervous system is concomitantlyreduced. By way of further example, the trauma or injury may also resultin the formation of a vaso-occlusion that restricts blood flow to thecentral nervous system.

Of course it is contemplated that the sirtuin activating compounds maybe employed to treat the central nervous system ischemic conditionirrespective of the cause of the condition. In one embodiment, theischemic condition results from a vaso-occlusion. The vaso-occlusion maybe any type of occlusion, but is typically a cerebral thrombosis or anembolism. In a further embodiment, the ischemic condition may resultfrom a hemorrhage. The hemorrhage may be any type of hemorrhage, but isgenerally a cerebral hemorrhage or a subararachnoid hemorrhage. In stillanother embodiment, the ischemic condition may result from the narrowingof a vessel. Generally speaking, the vessel may narrow as a result of avasoconstriction such as occurs during vasospasms, or due toarteriosclerosis. In yet another embodiment, the ischemic conditionresults from an injury to the brain or spinal cord.

In yet another aspect, a sirtuin activating compound may be administeredto reduce infarct size of the ischemic core following a central nervoussystem ischemic condition. Moreover, a sirtuin activating compound mayalso be beneficially administered to reduce the size of the ischemicpenumbra or transitional zone following a central nervous systemischemic condition.

In one embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of neurodegenerative disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more anti-neurodegeneration agents. For example, one or moresirtuin-activating compounds can be combined with an effective amount ofone or more of: L-DOPA; a dopamine agonist; an adenosine A_(2A) receptorantagonist; a COMT inhibitor; a MAO inhibitor; an N—NOS inhibitor; asodium channel antagonist; a selective N-methyl D-aspartate (NMDA)receptor antagonist; an AMPA/kainate receptor antagonist; a calciumchannel antagonist; a GABA-A receptor agonist; an acetyl-cholineesterase inhibitor; a matrix metalloprotease inhibitor; a PARPinhibitor; an inhibitor of p38 MAP kinase or c-jun-N-terminal kinases;TPA; NDA antagonists; beta-interferons; growth factors; glutamateinhibitors; and/or as part of a cell therapy.

Exemplary N—NOS inhibitors include4-(6-amino-pyridin-2-yl)-3-methoxyphenol6-[4-(2-dimethylamino-ethoxy)-2-methoxy-phenyl]-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2,3-dimet-hyl-phenyl]-pyridin-2-yl-amine,6-[4-(2-pyrrolidinyl-ethoxy)-2,3-dimethyl-p-henyl]-pyridin-2-yl-amine,6-[4-(4-(n-methyl)piperidinyloxy)-2,3-dimethyl-p-henyl]-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-3-methoxy-phenyl]-pyridin-2-yl-amine,6-[4-(2-pyrrolidinyl-ethoxy)-3-methoxy-phenyl]-pyridin-2-yl-amine,6-{4-[2-(6,7-dimethoxy-3,4-dihydro-1h-isoquinolin-2-yl)-ethoxy]-3-methoxy-phenyl}-pyridin-2-yl-amine,6-{3-methoxy-4-[2-(4-phenethyl-piper-azin-1-yl)-ethoxy]-phenyl}-pyridin-2-yl-amine,6-{3-methoxy-4-[2-(4-methyl-piperazin-1-yl)-ethoxy]-phenyl}-pyridin-2-yl-amine,6-{4-[2-(4-dimethylamin-o-piperidin-1-yl)-ethoxy]-3-methoxy-phenyl}-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-3-ethoxy-phenyl]-pyridin-2-yl-amine,6-[4-(2-pyrrolidinyl-ethoxy)-3-ethoxy-phenyl]-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2-isopropyl-phenyl]-pyridin-2-yl-amine,4-(6-amino-pyridin-yl)-3-cyclopropyl-phenol6-[2-cyclopropyl-4-(2-dimethyl-lamino-ethoxy)-phenyl]-pyridin-2-yl-amine,6-[2-cyclopropyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-pyridin-2-yl-amine,3-[3-(6-amino-pyridin-2-yl)-4-cycl-opropyl-phenoxy]-pyrrolidine-1-carboxylicacid tert-butyl ester6-[2-cyclopropyl-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,4-(6-amino-pyridin-2-yl)-3-cyclobutyl-phenol6-[2-cyclobutyl-4-(2-dime-thylamino-ethoxy)-phenyl]-pyridin-2-yl-amine,6-[2-cyclobutyl-4-(2-pyrrolid-in-1-yl-ethoxy)-phenyl]-pyridin-2-yl-amine,6-[2-cyclobutyl-4-(1-methyl-pyr-rolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,4-(6-amino-pyridin-2-yl)-3-cyclopentyl-phenol6-[2-cyclopentyl-4-(2-dimethylamino-ethoxy)-phenyl]-pyrid-in-2-yl-amine,6-[2-cyclopentyl-4-(2-pyrrolidin-1yl-ethoxy)-phenyl]-pyridin-2-yl-amine,3-[4-(6-amino-pyridin-2yl)-3-methoxy-phenoxy]-pyrrolidine-1-ca-rboxylicacid tert butyl ester6-[4-(1-methyl-pyrrolidin-3-yl-oxy)-2-metho-xy-phenyl]-pyridin-2-yl-amine,4-[4-(6-amino-pyridin-2yl)-3-methoxy-phenoxy-]-piperidine-1-carboxylicacid tert butyl ester6-[2-methoxy-4-(1-methyl-p-iperidin-4-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[4-(allyloxy)-2-methoxy-ph-enyl]-pyridin-2-yl-amine,4-(6-amino-pyridin-2-yl)-3-methoxy-6-allyl-phenol 12 and4-(6-amino-pyridin-2-yl)-3-methoxy-2-allyl-phenol 134-(6-amino-pyridin-2-yl)-3-methoxy-6-propyl-phenol6-[4-(2-dimethylamino-ethoxy)-2-methoxy-5-propyl-phenyl]-pyridin-yl-amine,6-[2-isopropyl-4-(pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropyl-4-(piperidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropyl-4-(1-methyl-azetidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropyl-4-(1-methyl-piperidin-4-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropyl-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amin-e6-[2-isopropyl-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropyl-4-(2-methyl-2-aza-bicyclo[2.2.1]hept-5-yl-oxy)-phenyl]-p-yridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2-methoxy-phenyl]-pyridin-2-yl-amine,6-{4-[2-(benzyl-methyl-amino)-ethoxy]-2-methoxy-phenyl}-pyridin-2-yl-amine,6-[2-methoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-pyridin-2-yl-amine,2-(6-amino-pyridin-2-yl)-5-(2-dimethylamino-ethoxy)-phenol2-[4-(6-amino-pyridin-2-yl)-3-methoxy-phenoxy]-acetamide6-[4-(2-amino-ethoxy)-2-methoxy-phenyl]-pyridin-2-yl-amine,6-{4-[2-(3,4-dihydro-1h-isoquinolin-2-yl)-ethoxy]-2-methoxy-phenyl}-pyrid-in-2-yl-amine,2-[4-(6-amino-pyridin-2-yl)-3-methoxy-phenoxy]-ethanol6-{2-methoxy-4-[2-(2,2,6,6-tetramethyl-piperidin-1-yl)-ethoxy]-phenyl}-py-ridin-2-yl-amine,6-{4-[2-(2,5-dimethyl-pyrrolidin-1-yl)-ethoxy]-2-methoxy-phenyl}-pyridin-2-yl-amine,6-{4-[2-(2,5-dimethyl-pyrrolidin-1-yl)-ethoxy]-2-methoxy-phenyl}-pyridin-2-yl-amine,2-[4-(6-amino-pyridin-2-yl)-3-methoxy-phenoxy]-1-(2,2,6,6-tetramethyl-piperidin-1-yl)-ethanone6-[2-methoxy-4-(1-methyl-pyrrolidin-2-yl-methoxy)-phenyl]-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2-propoxy-phenyl]-pyridin-2-yl-amine,6-{4-[2-(benzyl-methyl-amino)-ethoxy]-2-propoxy-phenyl}-pyridin-2-yl-amin-e6-[4-(2-ethoxy-ethoxy)-2-methoxy-phenyl]-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2-isopropoxy-phenyl]-pyridin-2-yl-amine,6-[4-(2-ethoxy-ethoxy)-2-isopropoxy-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(3-methyl-butoxy)-phenyl]-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2-ethoxy-phenyl]-pyridin-2-yl-amine,6-{4-[2-(benzyl-methyl-amino)-ethoxy]-2-ethoxy-phenyl}-pyridin-2-yl-amine,6-[2-ethoxy-4-(3-methyl-butoxy)-phenyl]-pyridin-2-yl-amine,1-(6-amino-3-aza-bicyclo[3.1.0]hex-3-yl)-2-[4-(6-amino-pyridin-2-yl)-3-et-hoxy-phenoxy]-ethanone6-[2-ethoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-py-ridin-2-yl-amine,3-{2-[4-(6-amino-pyridin-2-yl)-3-ethoxy-phenoxy]-ethyl}-3-aza-bicyclo[3.1.0]hex-6-yl-amine,1-(6-amino-3-aza-bicyclo[3.1.0]hex-3-yl)-2-[4-(6-amino-pyridin-2-yl)-3-methoxy-phenoxy]-ethanone3-{2-[4-(6-amino-pyridin-2-yl)-3-methoxy-phenoxy]-ethyl}-3-aza-bicyclo[3.-1.0]hex-6-yl-amine,6-[2-isopropoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-py-ridin-2-yl-amine,6-{4-[2-(benzyl-methyl-amino)-ethoxy]-2-isopropoxy-phenyl-}-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2-methoxy-5-propyl-phen-yl]-pyridin-2-yl-amine,6-[5-allyl-4-(2-dimethylamino-ethoxy)-2-methoxy-phe-nyl]-pyridin-2-yl-amine,6-[5-allyl-2-methoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-pyridin-2-yl-amine,6-[3-allyl-4-(2-dimethylamino-ethoxy)-2-methoxy-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(pyrrolidin-3-yl-oxy)-phenyl]-py-ridin-2-yl-amine,6-[2-methoxy-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-py-ridin-2-yl-amine,6-[2-ethoxy-4-(pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropoxy-4-(pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(piperidin-4-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(2,2,6,6-tetramethyl-piperidin-4-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropoxy-4-(pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,3-[4-(6-amino-pyridin-2-yl)-3-methoxy-phenoxy]-azetidine-1-carboxylicacid tert-butyl ester6-[4-(azetidin-3-yl-oxy)-2-methoxy-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(1-methyl-azetidin-3-yl-oxy)-phenyl]-pyridin-2-y-l-amine,6-[2-isopropoxy-4-(pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-isopropoxy-4-(pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[2-methoxy-4-(2-methyl-2-aza-bicyclo[2.2.1]hept-5-yl-oxy)-phenyl]-pyrid-in-2-yl-amine,6-[2-methoxy-4-(1-methyl-piperidin-4-yl-oxy)-phenyl]-pyridin-2-yl-amine,6-[4-(1-ethyl-piperidin-4-yl-oxy)-2-methoxy-phenyl]-pyridin-2-yl-amine,6-[5-allyl-2-methoxy-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-pyr-idin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2,6-dimethyl-phenyl]-pyridin-2-yl-amine,6-[2,6-dimethyl-4-(3-piperidin-1-yl-propoxy)-phenyl]-pyridin-2-yl-amine,6-[2,6-dimethyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-pyridin-2-y-l-amine,6-{2,6-dimethyl-4-[3-(4-methyl-piperazin-1-yl)-propoxy]-phenyl}-py-ridin-2-yl-amine,6-[2,6-dimethyl-4-(2-morpholin-4-yl-ethoxy)-phenyl]-pyrid-in-2-yl-amine,6-{4-[2-(benzyl-methyl-amino)-ethoxy]-2,6-dimethyl-phenyl}-p-yridin-2-yl-amine,2-[4-(6-amino-pyridin-2-yl)-3,5-dimethyl-phenoxy]-acetam-ide6-[4-(2-amino-ethoxy)-2,6-dimethyl-phenyl]-pyridin-2-yl-amine,6-[2-isopropyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-pyridin-2-yl-amine,2-(2,5-dimethyl-pyrrolidin-1-yl)-6-[2-isopropyl-4-(2-pyrrolidin-1-yl-etho-xy)-phenyl]-pyridine6-{4-[2-(3,5-dimethyl-piperidin-1-yl)-ethoxy]-2-isopr-opyl-phenyl}-pyridin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2-isopropyl-phenyl]-pyridin-2-yl-amine,6-[2-tert-butyl-4-(2-dimethylamino-ethoxy)-phen-yl-]-pyridin-2-yl-amine,6-[2-tert-butyl-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl-]-pyridin-2-yl-amine,6-[4-(2-pyrrolidinyl-ethoxy)-2,5-dimethyl-phenyl]-pyr-idin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-2,5-dimethyl-phenyl]-pyridin-2-yl-amine,6-[4-(2-(4-phenethylpiperazin-1-yl)-ethoxy)-2,5-dimethyl-pheny-l]-pyridin-2-yl-amine,6-[2-cyclopropyl-4-(2-dimethylamino-1-methyl-ethoxy)-phenyl]-pyridin-2-yl-amine,6-[cyclobutyl-4-(2-dimethylamino-1-methyl-etho-xy)-phenyl]-pyridin-2-yl-amine,6-[4-(allyloxy)-2-cyclobutyl-phenyl]-pyridi-n-2-ylamine,2-allyl-4-(6-amino-pyridin-2-yl)-3-cyclobutyl-phenol and2-allyl-4-(6-amino-pyridin-2-yl)-5-cyclobutyl-phenol4-(6-amino-pyridin-2-yl)-5-cyclobutyl-2-propyl-phenol4-(6-amino-pyridin-2-yl)-3-cyclobutyl-2-propyl-phenol6-[2-cyclobutyl-4-(2-dimethylamino-1-methyl-ethoxy)-5-propyl-phenyl]-pyri-din-2-yl-amine,6-[2-cyclobutyl-4-(2-dimethylamino-1-methyl-ethoxy)-3-propy-l-phenyl]-pyridin-2-yl-amine,6-[2-cyclobutyl-4-(2-dimethylamino-ethoxy)-5-propyl-phenyl]-pyridin-2-yl-amine,6-[2-cyclobutyl-4-(2-dimethylamino-ethox-y)-3-propyl-phenyl]-pyridin-2-yl-amine,6-[2-cyclobutyl-4-(1-methyl-pyrroli-din-3-yl-oxy)-5-propyl-phenyl]-pyridin-2-yl-amine,6-[cyclobutyl-4-(1-methyl-1-pyrrolidin-3-yl-oxy)-3-propyl-phenyl]-pyridin-2-yl-amine,2-(4-benzyloxy-5-hydroxy-2-methoxy-phenyl)-6-(2,5-dimethyl-pyrrol-1-yl)-p-yridine6-[4-(2-dimethylamino-ethoxy)-5-ethoxy-2-methoxy-phenyl]-pyridin-2-yl-amine,6-[5-ethyl-2-methoxy-4-(1-methyl-piperidin-4-yl-oxy)-phenyl]-pyr-idin-2-yl-amine,6-[5-ethyl-2-methoxy-4-(piperidin-4-yl-oxy)-phenyl]-pyridi-n-2-yl-amine,6-[2,5-dimethoxy-4-(1-methyl-pyrrolidin-3-yl-oxy)-phenyl]-pyr-idin-2-yl-amine,6-[4-(2-dimethylamino-ethoxy)-5-ethyl-2-methoxy-phenyl]-py-ridin-2-yl-amine.

Exemplary NMDA receptor antagonist include(+)-(1S,2S)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-pro-panol,(1S,2S)-1-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-4-phenylpiperi-dino)-1-propanol,(3R,4S)-3-(4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl-)-chroman-4,7-diol,(1R*,2R*)-1-(4-hydroxy-3-methylphenyl)-2-(4-(4-fluoro-phenyl)-4-hydroxypiperidin-1-yl)-propan-1-ol-mesylateor a pharmaceutically acceptable acid addition salt thereof.

Exemplary dopamine agonist include ropininole; L-dopa decarboxylaseinhibitors such as carbidopa or benserazide, bromocriptine,dihydroergocryptine, etisulergine, AF-14, alaptide, pergolide,piribedil; dopamine D1 receptor agonists such as A-68939, A-77636,dihydrexine, and SKF-38393; dopamine D2 receptor agonists such ascarbergoline, lisuride, N-0434, naxagolide, PD-118440, pramipexole,quinpirole and ropinirole; dopamine/β-adrenegeric receptor agonists suchas DPDMS and dopexamine; dopamine/5-HT uptake inhibitor/5-HT-1A agonistssuch as roxindole; dopamine/opiate receptor agonists such as NIH-10494;α2-adrenergic antagonist/dopamine agonists such as terguride;α2-adrenergic antagonist/dopamine D2 agonists such as ergolines andtalipexole; dopamine uptake inhibitors such as GBR-12909, GBR-13069,GYKI-52895, and NS-2141; monoamine oxidase-B inhibitors such asselegiline, N-(2-butyl)-N-methylpropargylamine,N-methyl-N-(2-pentyl)propargylamine, AGN-1133, ergot derivatives,lazabemide, LU-53439, MD-280040 and mofegiline; and COMT inhibitors suchas CGP-28014.

Exemplary acetyl cholinesterase inhibitors include donepizil,1-(2-methyl-1H-benzimida-zol-5-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(2-phenyl-1H-benzimidazol-5-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-pr-opanone;1-(1-ethyl-2-methyl-1H-benzimidazol-5-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(2-methyl-6-benzothiazolyl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(2-methyl-6-benzothiazolyl)-3-[1-[(2-methyl-4-thiazolyl)methyl]-4-piperidinyl]-1-propanone;1-(5-methyl-benzo[b]thie-n-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(6-methyl-benzo[b]thien-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-prop-anone;1-(3,5-dimethyl-benzo[b]thien-2-yl)-3-[1-(phenylmethyl)-4-piperidin-yl]-1-propanone;1-(benzo[b]thien-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(benzofuran-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-pro-panone;1-(1-phenylsulfonyl-6-methyl-indol-2-yl)-3-[1-(phenylmethyl)-4-pip-eridinyl]-1-propanone;1-(6-methyl-indol-2-yl)-3-[1-(phenylmethyl)-4-piper-idinyl]-1-propanone;1-(1-phenylsulfonyl-5-amino-indol-2-yl)-3-[1-(phenylm-ethyl)-4-piperidinyl]-1-propanone;1-(5-amino-indol-2-yl)-3-[1-(phenylmet-hyl)-4-piperidinyl]-1-propanone;and1-(5-acetylamino-indol-2-yl)-3-[1-(ph-enylmethyl)-4-piperidinyl]-1-propanone.1-(6-quinolyl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(5-indolyl)-3-[1-(phenylmethyl)-4-piperidiny-l]-1-propanone;1-(5-benzthienyl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-pro-panone;1-(6-quinazolyl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(6-benzoxazolyl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(5-benzofuranyl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(5-methyl-benzimidazol-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propa-none;1-(6-methyl-benzimidazol-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(5-chloro-benzo[b]thien-2-yl)-3-[1-(phenylmethyl)-4-piperidin-yl]-1-propanone;1-(5-azaindol-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-p-ropanone;1-(6-azabenzo[b]thien-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(1H-2-oxo-pyrrolo[2′,3′,5,6]benzo[b]thieno-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(6-methyl-benzothiazol-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(6-methoxy-indol-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-propanone;1-(6-methoxy-benzo[b]thien-2-yl)-3-[1-(phenylmethyl)-4-piperidinyl]-1-pro-panone;1-(6-acetylamino-benzo[b]thien-2-yl)-3-[1-(phenylmethyl)-4-piperid-inyl]-1-propanone;1-(5-acetylamino-benzo[b]thien-2-yl)-3-[1-(phenylmethyl-)-4-piperidinyl]-1-propanone;6-hydroxy-3-[2-[1-(phenylmethyl)-4-piperidin-yl]ethyl]-1,2-benzisoxazole;5-methyl-3-[2-[1-(phenylmethyl)-4-piperidinyl-]ethyl]-1,2-benzisoxazole;6-methoxy-3-[2-[1(phenylmethyl)-4-piperidinyl]et-hyl]-1,2-benzisoxazole;6-acetamide-3-[2-[1-(phenylmethyl)-4-piperidinyl]-ethyl]-1,2-benzisoxazole;6-amino-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl-1]-1,2-benzisoxazole;6-(4-morpholinyl)-3-[2-[1-(phenylmethyl)-4-piperidin-yl]ethyl]-1,2-benzisoxazole;5,7-dihydro-3-[2-[1-(phenylmethyl)-4-piperidi-nyl]ethyl]-6H-pyrrolo[4,5-f]-1,2-benzisoxazol-6-one;3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-1,2-benzisothiazole;3-[2-[1-(phenylmethyl)-4-piperidinyl]ethenyl]-1,2-benzisoxazole;6-phenylamino-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-1,-2,-benzisoxaz-ole;6-(2-thiazoly)-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-1,2-benzis-oxazole;6-(2-oxazolyl)-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-1,2-benzisoxazole;6-pyrrolidinyl-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-1,-2-benzisoxazole;5,7-dihydro-5,5-dimethyl-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H-pyrrolo[4,5-f]-1,2-benzisoxazole-6-one;6,8-dihydro-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-7H-pyrrolo[5,4-g]-1,2-benzisoxazole-7-one;3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-5,6,-8-trihydro-7H-isoxazolo[4,5-g]-quinolin-7-one;1-benzyl-4-((5,6-dimethoxy-1-indanon)-2-yl)methylpiperidine,1-benzyl-4-((5,6-dimethoxy-1-indanon)-2-ylidenyl)methylpiperidine,1-benzyl-4-((5-methoxy-1-indanon)-2-yl)methylp-iperidine,1-benzyl-4-((5,6-diethoxy-1-indanon)-2-yl)methylpiperidine,1-benzyl-4-((5,6-methylenedioxy-1-indanon)-2-yl)methylpiperidine,1-(m-nitrobenzyl)-4-((5,6-dimethoxy-1-indanon)-2-yl)methylpiperidine,1-cyclohexymethyl-4-((5,6-dimethoxy-1-indanon)-2-yl)methylpiperidine,1-(m-florobenzyl)-4-((5,6-dimethoxy-1-indanon)-2-yl)methylpiperidine,1-benzyl-4-((5,6-dimethoxy-1-indanon)-2-yl)propylpiperidine, and1-benzyl-4-((5-isopropoxy-6-methoxy-1-indanon)-2-yl)methylpiperidine.

Exemplary calcium channel antagonists include diltiazem, omega-conotoxinGVIA, methoxyverapamil, amlodipine, felodipine, lacidipine, andmibefradil.

Exemplary GABA-A receptor modulators include clomethiazole; IDDB;gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol); ganaxolone(3α-hydroxy-3β-methyl-5α-pregnan-20-one); fengabine(2-[(butylimino)-(2-chlorophenyl)methyl]-4-chlorophenol);2-(4-methoxyphenyl)-2,5,6,7,8,9-hexahydro-pyrazolo[4,3-c]cinnolin-3-one;7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-phenyl-1,2,4-triazolo[4,3-b]pyridazine;(3-fluoro-4-methylphenyl)-N-({-1-[(2-methylphenyl)methyl]-benzimidazol-2-yl}methyl)-N-pentylcarboxamide;and 3-(aminomethyl)-5-methylhexanoic acid.

Exemplary potassium channel openers include diazoxide, flupirtine,pinacidil, levcromakalim, rilmakalim, chromakalim, PCO-400 and SKP-450(2-[2″(1″,3″-dioxolone)-2-methyl]-4-(2′-oxo-1′-pyrrolidinyl)-6-nitro-2H-1-benzopyra-n).

Exemplary AMPA/kainate receptor antagonists include6-cyano-7-nitroquinoxalin-2,3-di-one (CNQX);6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX);6,7-dinitroquinoxaline-2,3-dione (DNQX);1-(4-aminophenyl)-4-methyl-7,8-m-ethylenedioxy-5H-2,3-benzodiazepinehydrochloride; and2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline.

Exemplary sodium channel antagonists include ajmaline, procainamide,flecainide and riluzole.

Exemplary matrix-metalloprotease inhibitors include4-[4-(4-fluorophenoxy)benzenesulfon-ylamino]tetrahydropyran-4-carboxylicacid hydroxyamide;5-Methyl-5-(4-(4′-fluorophenoxy)-phenoxy)-pyrimidine-2,4,6-trione;5-n-Butyl-5-(4-(4′-fluorophenoxy)-phenoxy)-pyrimidine-2,4,6-trione andprinomistat.

Poly(ADP ribose) polymerase (PARP) is an abundant nuclear enzyme whichis activated by DNA strand single breaks to synthesize poly (ADP ribose)from NAD. Under normal conditions, PARP is involved in base excisionrepair caused by oxidative stress via the activation and recruitment ofDNA repair enzymes in the nucleus. Thus, PARP plays a role in cellnecrosis and DNA repair. PARP also participates in regulating cytokineexpression that mediates inflammation. Under conditions where DNA damageis excessive (such as by acute excessive exposure to a pathologicalinsult), PARP is over-activated, resulting in cell-based energeticfailure characterized by NAD depletion and leading to ATP consumption,cellular necrosis, tissue injury, and organ damage/failure. PARP isthought to contribute to neurodegeneration by depleting nicotinamideadenine dinucleotide (NAD+) which then reduces adenosine triphosphate(ATP; Cosi and Marien, Ann. N.Y. Acad. Sci., 890:227, 1999) contributingto cell death which can be prevented by PARP inhibitors. Exemplory PARPinhibitors can be found in Southan and Szabo, Current MedicinalChemistry, 10:321, 2003.

Exemplary inhibitors of p38 MAP kinase and c-jun-N-terminal kinasesinclude pyridyl imidazoles, such as PD 169316, isomeric PD 169316, SB203580, SB 202190, SB 220026, and RWJ 67657. Others are described inU.S. Pat. No. 6,288,089, and incorporated by reference herein.

In an exemplary embodiment, a combination therapy for treating orpreventing MS comprises a therapeutically effective amount of one ormore sirtuin-modulating compounds that increase the level and/oractivity of a sirtuin protein and one or more of Avonex® (interferonbeta-1a), Tysabri® (natalizumab), or Fumaderm® (BG-12/Oral Fumarate).

In another embodiment, a combination therapy for treating or preventingdiabetic neuropathy or conditions associated therewith comprises atherapeutically effective amount of one or more sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinand one or more of tricyclic antidepressants (TCAs) (including, forexample, imipramine, amytriptyline, desipramine and nortriptyline),serotonin reuptake inhibitors (SSRIs) (including, for example,fluoxetine, paroxetine, sertralene, and citalopram) and antiepilepticdrugs (AEDs) (including, for example, gabapentin, carbamazepine, andtopimirate).

In another embodiment, the invention provides a method for treating orpreventing a polyglutamine disease using a combination comprising atleast one sirtuin activating compound and at least one HDAC I/IIinhibitor. Examples of HDAC I/II inhibitors include hydroxamic acids,cyclic peptides, benzamides, short-chain fatty acids, and depudecin.

Examples of hydroxamic acids and hydroxamic acid derivatives, but arenot limited to, trichostatin A (TSA), suberoylanilide hydroxamic acid(SAHA), oxamflatin, suberic bishydroxamic acid (SBHA),m-carboxy-cinnamic acid bishydroxamic acid (CBHA), valproic acid andpyroxamide. TSA was isolated as an antifungi antibiotic (Tsuji et al(1976) J. Antibiot (Tokyo) 29:1-6) and found to be a potent inhibitor ofmammalian HDAC (Yoshida et al. (1990) J. Biol. Chem. 265:17174-17179).The finding that TSA-resistant cell lines have an altered HDAC evidencesthat this enzyme is an important target for TSA. Other hydroxamicacid-based HDAC inhibitors, SAHA, SBHA, and CBHA are synthetic compoundsthat are able to inhibit HDAC at micromolar concentration or lower invitro or in vivo. Glick et al. (1999) Cancer Res. 59:43924399. Thesehydroxamic acid-based HDAC inhibitors all possess an essentialstructural feature: a polar hydroxamic terminal linked through ahydrophobic methylene spacer (e.g. 6 carbon at length) to another polarsite which is attached to a terminal hydrophobic moiety (e.g., benzenering). Compounds developed having such essential features also fallwithin the scope of the hydroxamic acids that may be used as HDACinhibitors.

Cyclic peptides used as HDAC inhibitors are mainly cyclic tetrapeptides.Examples of cyclic peptides include, but are not limited to, trapoxin A,apicidin and depsipeptide. Trapoxin A is a cyclic tetrapeptide thatcontains a 2-amino-8-oxo-9,10-epoxy-decanoyl (AOE) moiety. Kijima et al.(1993) J. Biol. Chem. 268:22429-22435. Apicidin is a fungal metabolitethat exhibits potent, broad-spectrum antiprotozoal activity and inhibitsHDAC activity at nanomolar concentrations. Darkin-Rattray et al. (1996)Proc. Natl. Acad. Sci. USA. 93; 13143-13147. Depsipeptide is isolatedfrom Chromobacterium violaceum, and has been shown to inhibit HDACactivity at micromolar concentrations.

Examples of benzamides include but are not limited to MS-27-275. Saitoet al. (1990) Proc. Natl. Acad. Sci. USA. 96:4592-4597. Examples ofshort-chain fatty acids include but are not limited to butyrates (e.g.,butyric acid, arginine butyrate and phenylbutyrate (PB)). Newmark et al.(1994) Cancer Lett. 78:1-5; and Carducci et al. (1997) Anticancer Res.17:3972-3973. In addition, depudecin which has been shown to inhibitHDAC at micromolar concentrations (Kwon et al. (1998) Proc. Natl. Acad.Sci. USA. 95:3356-3361) also falls within the scope of histonedeacetylase inhibitor as described herein.

Blood Coagulation Disorders

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or preventblood coagulation disorders (or hemostatic disorders). As usedinterchangeably herein, the terms “hemostasis”, “blood coagulation,” and“blood clotting” refer to the control of bleeding, including thephysiological properties of vasoconstriction and coagulation. Bloodcoagulation assists in maintaining the integrity of mammaliancirculation after injury, inflammation, disease, congenital defect,dysfunction or other disruption. After initiation of clotting, bloodcoagulation proceeds through the sequential activation of certain plasmaproenzymes to their enzyme forms (see, for example, Coleman, R. W. etal. (eds.) Hemostasis and Thrombosis, Second Edition, (1987)). Theseplasma glycoproteins, including Factor XII, Factor XI, Factor IX, FactorX, Factor VII, and prothrombin, are zymogens of serine proteases. Mostof these blood clotting enzymes are effective on a physiological scaleonly when assembled in complexes on membrane surfaces with proteincofactors such as Factor VIII and Factor V. Other blood factors modulateand localize clot formation, or dissolve blood clots. Activated proteinC is a specific enzyme that inactivates procoagulant components. Calciumions are involved in many of the component reactions. Blood coagulationfollows either the intrinsic pathway, where all of the proteincomponents are present in blood, or the extrinsic pathway, where thecell-membrane protein tissue factor plays a critical role. Clotformation occurs when fibrinogen is cleaved by thrombin to form fibrin.Blood clots are composed of activated platelets and fibrin.

Further, the formation of blood clots does not only limit bleeding incase of an injury (hemostasis), but may lead to serious organ damage anddeath in the context of atherosclerotic diseases by occlusion of animportant artery or vein. Thrombosis is thus blood clot formation at thewrong time and place. It involves a cascade of complicated and regulatedbiochemical reactions between circulating blood proteins (coagulationfactors), blood cells (in particular platelets), and elements of aninjured vessel wall.

Accordingly, the present invention provides anticoagulation andantithrombotic treatments aiming at inhibiting the formation of bloodclots in order to prevent or treat blood coagulation disorders, such asmyocardial infarction, stroke, loss of a limb by peripheral arterydisease or pulmonary embolism.

As used interchangeably herein, “modulating or modulation of hemostasis”and “regulating or regulation of hemostasis” includes the induction(e.g., stimulation or increase) of hemostasis, as well as the inhibition(e.g., reduction or decrease) of hemostasis.

In one aspect, the invention provides a method for reducing orinhibiting hemostasis in a subject by administering a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.The compositions and methods disclosed herein are useful for thetreatment or prevention of thrombotic disorders. As used herein, theterm “thrombotic disorder” includes any disorder or conditioncharacterized by excessive or unwanted coagulation or hemostaticactivity, or a hypercoagulable state. Thrombotic disorders includediseases or disorders involving platelet adhesion and thrombusformation, and may manifest as an increased propensity to formthromboses, e.g., an increased number of thromboses, thrombosis at anearly age, a familial tendency towards thrombosis, and thrombosis atunusual sites. Examples of thrombotic disorders include, but are notlimited to, thromboembolism, deep vein thrombosis, pulmonary embolism,stroke, myocardial infarction, miscarriage, thrombophilia associatedwith anti-thrombin III deficiency, protein C deficiency, protein Sdeficiency, resistance to activated protein C, dysfibrinogenemia,fibrinolytic disorders, homocystinuria, pregnancy, inflammatorydisorders, myeloproliferative disorders, arteriosclerosis, angina, e.g.,unstable angina, disseminated intravascular coagulation, thromboticthrombocytopenic purpura, cancer metastasis, sickle cell disease,glomerular nephritis, and drug induced thrombocytopenia (including, forexample, heparin induced thrombocytopenia). In addition,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to prevent thrombotic events or toprevent re-occlusion during or after therapeutic clot lysis orprocedures such as angioplasty or surgery.

In another embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of blood coagulation disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinand one or more anti-coagulation or anti-thrombosis agents. For example,one or more sirtuin-modulating compounds can be combined with aneffective amount of one or more of: aspirin, heparin, and oral Warfarinthat inhibits Vit K-dependent factors, low molecular weight heparinsthat inhibit factors X and II, thrombin inhibitors, inhibitors ofplatelet GP IIbIIIa receptors, inhibitors of tissue factor (TF),inhibitors of human von Willebrand factor, inhibitors of one or morefactors involved in hemostasis (in particular in the coagulationcascade). In addition, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can be combined withthrombolytic agents, such as t-PA, streptokinase, reptilase, TNK-t-PA,and staphylokinase.

Weight Control

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing weight gain or obesity in a subject. For example,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used, for example, to treat or preventhereditary obesity, dietary obesity, hormone related obesity, obesityrelated to the administration of medication, to reduce the weight of asubject, or to reduce or prevent weight gain in a subject. A subject inneed of such a treatment may be a subject who is obese, likely to becomeobese, overweight, or likely to become overweight. Subjects who arelikely to become obese or overweight can be identified, for example,based on family history, genetics, diet, activity level, medicationintake, or various combinations thereof.

In yet other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered tosubjects suffering from a variety of other diseases and conditions thatmay be treated or prevented by promoting weight loss in the subject.Such diseases include, for example, high blood pressure, hypertension,high blood cholesterol, dyslipidemia, type 2 diabetes, insulinresistance, glucose intolerance, hyperinsulinemia, coronary heartdisease, angina pectoris, congestive heart failure, stroke, gallstones,cholescystitis and cholelithiasis, gout, osteoarthritis, obstructivesleep apnea and respiratory problems, some types of cancer (such asendometrial, breast, prostate, and colon), complications of pregnancy,poor female reproductive health (such as menstrual irregularities,infertility, irregular ovulation), bladder control problems (such asstress incontinence); uric acid nephrolithiasis; psychological disorders(such as depression, eating disorders, distorted body image, and lowself esteem). Stunkard A J, Wadden T A. (Editors) Obesity: theory andtherapy, Second Edition. New York: Raven Press, 1993. Finally, patientswith AIDS can develop lipodystrophy or insulin resistance in response tocombination therapies for AIDS.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for inhibitingadipogenesis or fat cell differentiation, whether in vitro or in vivo.In particular, high circulating levels of insulin and/or insulin likegrowth factor (IGF) 1 will be prevented from recruiting preadipocytes todifferentiate into adipocytes. Such methods may be used for treating orpreventing obesity.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for reducingappetite and/or increasing satiety, thereby causing weight loss oravoidance of weight gain. A subject in need of such a treatment may be asubject who is overweight, obese or a subject likely to becomeoverweight or obese. The method may comprise administering daily or,every other day, or once a week, a dose, e.g., in the form of a pill, toa subject. The dose may be an “appetite reducing dose.”

In other embodiments, a sirtuin-modulating compound that decreases thelevel and/or activity of a sirtuin protein may be used to stimulateappetite and/or weight gain. A method may comprise administering to asubject, such as a subject in need thereof, a pharmaceutically effectiveamount of a sirtuin-modulating agent that decreases the level and/oractivity of a sirtuin protein, such as SIRT1 and/or SIRT3. A subject inneed of such a treatment may be a subject who has cachexia or may belikely to develop cachexia. A combination of agents may also beadministered. A method may further comprise monitoring in the subjectthe state of the disease or of activation of sirtuins, for example, inadipose tissue.

Methods for stimulating fat accumulation in cells may be used in vitro,to establish cell models of weight gain, which may be used, e.g., foridentifying other drugs that prevent weight gain.

Also provided are methods for modulating adipogenesis or fat celldifferentiation, whether in vitro or in vivo. In particular, highcirculating levels of insulin and/or insulin like growth factor (IGF) 1will be prevented from recruiting preadipocytes to differentiate intoadipocytes. Such methods may be used to modulate obesity. A method forstimulating adipogenesis may comprise contacting a cell with asirtuin-modulating agent that decreases the level and/or activity of asirtuin protein.

In another embodiment, the invention provides methods of decreasing fator lipid metabolism in a subject by administering a sirtuin-modulatingcompound that decreases the level and/or activity of a sirtuin protein.The method includes administering to a subject an amount of asirtuin-modulating compound, e.g., in an amount effective to decreasemobilization of fat to the blood from WAT cells and/or to decrease fatburning by BAT cells.

Methods for promoting appetite and/or weight gain may include, forexample, prior identifying a subject as being in need of decreased fator lipid metabolism, e.g., by weighing the subject, determining the BMIof the subject, or evaluating fat content of the subject or sirtuinactivity in cells of the subject. The method may also include monitoringthe subject, e.g., during and/or after administration of asirtuin-modulating compound. The administering can include one or moredosages, e.g., delivered in boluses or continuously. Monitoring caninclude evaluating a hormone or a metabolite. Exemplary hormones includeleptin, adiponectin, resistin, and insulin. Exemplary metabolitesinclude triglyercides, cholesterol, and fatty acids.

In one embodiment, a sirtuin-modulating compound that decreases thelevel and/or activity of a sirtuin protein may be used to modulate(e.g., increase) the amount of subcutaneous fat in a tissue, e.g., infacial tissue or in other surface-associated tissue of the neck, hand,leg, or lips. The sirtuin-modulating compound may be used to increasethe rigidity, water retention, or support properties of the tissue. Forexample, the sirtuin-modulating compound can be applied topically, e.g.,in association with another agent, e.g., for surface-associated tissuetreatment. The sirtuin-modulating compound may also be injectedsubcutaneously, e.g., within the region where an alteration insubcutaneous fat is desired.

A method for modulating weight may further comprise monitoring theweight of the subject and/or the level of modulation of sirtuins, forexample, in adipose tissue.

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing weight gain or obesity.For example, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-obesity agents. Exemplary anti-obesityagents include, for example, phenylpropanolamine, ephedrine,pseudoephedrine, phentermine, a cholecystokinin-A agonist, a monoaminereuptake inhibitor (such as sibutramine), a sympathomimetic agent, aserotonergic agent (such as dexfenfluramine or fenfluramine), a dopamineagonist (such as bromocriptine), a melanocyte-stimulating hormonereceptor agonist or mimetic, a melanocyte-stimulating hormone analog, acannabinoid receptor antagonist, a melanin concentrating hormoneantagonist, the OB protein (leptin), a leptin analog, a leptin receptoragonist, a galanin antagonist or a GI lipase inhibitor or decreaser(such as orlistat). Other anorectic agents include bombesin agonists,dehydroepiandrosterone or analogs thereof, glucocorticoid receptoragonists and antagonists, orexin receptor antagonists, urocortin bindingprotein antagonists, agonists of the glucagon-like peptide-1 receptorsuch as Exendin and ciliary neurotrophic factors such as Axokine.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered to reducedrug-induced weight gain. For example, a sirtuin-modulating compoundthat increases the level and/or activity of a sirtuin protein may beadministered as a combination therapy with medications that maystimulate appetite or cause weight gain, in particular, weight gain dueto factors other than water retention. Examples of medications that maycause weight gain, include for example, diabetes treatments, including,for example, sulfonylureas (such as glipizide and glyburide),thiazolidinediones (such as pioglitazone and rosiglitazone),meglitinides, nateglinide, repaglinide, sulphonylurea medicines, andinsulin; anti-depressants, including, for example, tricyclicantidepressants (such as amitriptyline and imipramine), irreversiblemonoamine oxidase inhibitors (MAOIs), selective serotonin reuptakeinhibitors (SSRIs), bupropion, paroxetine, and mirtazapine; steroids,such as, for example, prednisone; hormone therapy; lithium carbonate;valproic acid; carbamazepine; chlorpromazine; thiothixene; beta blockers(such as propranolo); alpha blockers (such as clonidine, prazosin andterazosin); and contraceptives including oral contraceptives (birthcontrol pills) or other contraceptives containing estrogen and/orprogesterone (Depo-Provera, Norplant, Ortho), testosterone or Megestrol.In another exemplary embodiment, sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein may beadministered as part of a smoking cessation program to prevent weightgain or reduce weight already gained.

Metabolic Disorders/Diabetes

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing a metabolic disorder, such as insulin-resistance, apre-diabetic state, type II diabetes, and/or complications thereof.Administration of a sirtuin-modulating compounds that increases thelevel and/or activity of a sirtuin protein may increase insulinsensitivity and/or decrease insulin levels in a subject. A subject inneed of such a treatment may be a subject who has insulin resistance orother precursor symptom of type II diabetes, who has type II diabetes,or who is likely to develop any of these conditions. For example, thesubject may be a subject having insulin resistance, e.g., having highcirculating levels of insulin and/or associated conditions, such ashyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired glucosetolerance, high blood glucose sugar level, other manifestations ofsyndrome X, hypertension, atherosclerosis and lipodystrophy.

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing a metabolic disorder. Forexample, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-diabetic agents. Exemplaryanti-diabetic agents include, for example, an aldose reductaseinhibitor, a glycogen phosphorylase inhibitor, a sorbitol dehydrogenaseinhibitor, a protein tyrosine phosphatase 1B inhibitor, a dipeptidylprotease inhibitor, insulin (including orally bioavailable insulinpreparations), an insulin mimetic, metformin, acarbose, a peroxisomeproliferator-activated receptor-γ (PPAR-γ) ligand such as troglitazone,rosaglitazone, pioglitazone or GW-1929, a sulfonylurea, glipazide,glyburide, or chlorpropamide wherein the amounts of the first and secondcompounds result in a therapeutic effect. Other anti-diabetic agentsinclude a glucosidase inhibitor, a glucagon-like peptide-1 (GLP-1),insulin, a PPAR α/γ dual agonist, a meglitimide and an αP2 inhibitor. Inan exemplary embodiment, an anti-diabetic agent may be a dipeptidylpeptidase IV (DP-IV or DPP-IV) inhibitor, such as, for example LAF237from Novartis (NVP DPP728;1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine)or MK-04301 from Merck (see e.g., Hughes et al., Biochemistry 38:11597-603 (1999)).

Inflammatory Diseases

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or prevent adisease or disorder associated with inflammation. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered prior to the onset of, at, or after the initiationof inflammation. When used prophylactically, the compounds arepreferably provided in advance of any inflammatory response or symptom.Administration of the compounds may prevent or attenuate inflammatoryresponses or symptoms.

Exemplary inflammatory conditions include, for example, multiplesclerosis, rheumatoid arthritis, psoriatic arthritis, degenerative jointdisease, spondouloarthropathies, gouty arthritis, systemic lupuserythematosus, juvenile arthritis, rheumatoid arthritis, osteoarthritis,osteoporosis, diabetes (e.g., insulin dependent diabetes mellitus orjuvenile onset diabetes), menstrual cramps, cystic fibrosis,inflammatory bowel disease, irritable bowel syndrome, Crohn's disease,mucous colitis, ulcerative colitis, gastritis, esophagitis,pancreatitis, peritonitis, Alzheimer's disease, shock, ankylosingspondylitis, gastritis, conjunctivitis, pancreatis (acute or chronic),multiple organ injury syndrome (e.g., secondary to septicemia ortrauma), myocardial infarction, atherosclerosis, stroke, reperfusioninjury (e.g., due to cardiopulmonary bypass or kidney dialysis), acuteglomerulonephritis, vasculitis, thermal injury (i.e., sunburn),necrotizing enterocolitis, granulocyte transfusion associated syndrome,and/or Sjogren's syndrome. Exemplary inflammatory conditions of the skininclude, for example, eczema, atopic dermatitis, contact dermatitis,urticaria, schleroderma, psoriasis, and dermatosis with acuteinflammatory components.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to treat orprevent allergies and respiratory conditions, including asthma,bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity,emphysema, chronic bronchitis, acute respiratory distress syndrome, andany chronic obstructive pulmonary disease (COPD). The compounds may beused to treat chronic hepatitis infection, including hepatitis B andhepatitis C.

Additionally, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to treat autoimmunediseases and/or inflammation associated with autoimmune diseases such asorgan-tissue autoimmune diseases (e.g., Raynaud's syndrome),scleroderma, myasthenia gravis, transplant rejection, endotoxin shock,sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmunethyroiditis, uveitis, systemic lupus erythematosis, Addison's disease,autoimmune polyglandular disease (also known as autoimmune polyglandularsyndrome), and Grave's disease.

In certain embodiments, one or more sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein may be takenalone or in combination with other compounds useful for treating orpreventing inflammation. Exemplary anti-inflammatory agents include, forexample, steroids (e.g., cortisol, cortisone, fludrocortisone,prednisone, 6α-methylprednisone, triamcinolone, betamethasone ordexamethasone), nonsteroidal antiinflammatory drugs (NSAIDS (e.g.,aspirin, acetaminophen, tolmetin, ibuprofen, mefenamic acid, piroxicam,nabumetone, rofecoxib, celecoxib, etodolac or nimesulide). In anotherembodiment, the other therapeutic agent is an antibiotic (e.g.,vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime,ceftriaxone, cefixime, rifampinmetronidazole, doxycycline orstreptomycin). In another embodiment, the other therapeutic agent is aPDE4 inhibitor (e.g., roflumilast or rolipram). In another embodiment,the other therapeutic agent is an antihistamine (e.g., cyclizine,hydroxyzine, promethazine or diphenhydramine). In another embodiment,the other therapeutic agent is an anti-malarial (e.g., artemisinin,artemether, artsunate, chloroquine phosphate, mefloquine hydrochloride,doxycycline hyclate, proguanil hydrochloride, atovaquone orhalofantrine). In one embodiment, the other therapeutic agent isdrotrecogin alfa.

Further examples of anti-inflammatory agents include, for example,aceclofenac, acemetacin, e-acetamidocaproic acid, acetaminophen,acetaminosalol, acetanilide, acetylsalicylic acid, S-adenosylmethionine,alclofenac, alclometasone, alfentanil, algestone, allylprodine,alminoprofen, aloxiprin, alphaprodine, aluminum bis(acetylsalicylate),amcinonide, amfenac, aminochlorthenoxazin, 3-amino-4-hydroxybutyricacid, 2-amino-4-picoline, aminopropylon, aminopyrine, amixetrine,ammonium salicylate, ampiroxicam, amtolmetin guacil, anileridine,antipyrine, antrafenine, apazone, beclomethasone, bendazac, benorylate,benoxaprofen, benzpiperylon, benzydamine, benzylmorphine, bermoprofen,betamethasone, betamethasone-17-valerate, bezitramide, α-bisabolol,bromfenac, p-bromoacetanilide, 5-bromosalicylic acid acetate,bromosaligenin, bucetin, bucloxic acid, bucolome, budesonide, bufexamac,bumadizon, buprenorphine, butacetin, butibufen, butorphanol,carbamazepine, carbiphene, carprofen, carsalam, chlorobutanol,chloroprednisone, chlorthenoxazin, choline salicylate, cinchophen,cinmetacin, ciramadol, clidanac, clobetasol, clocortolone, clometacin,clonitazene, clonixin, clopirac, cloprednol, clove, codeine, codeinemethyl bromide, codeine phosphate, codeine sulfate, cortisone,cortivazol, cropropamide, crotethamide, cyclazocine, deflazacort,dehydrotestosterone, desomorphine, desonide, desoximetasone,dexamethasone, dexamethasone-21-isonicotinate, dexoxadrol,dextromoramide, dextropropoxyphene, deoxycorticosterone, dezocine,diampromide, diamorphone, diclofenac, difenamizole, difenpiramide,diflorasone, diflucortolone, diflunisal, difluprednate, dihydrocodeine,dihydrocodeinone enol acetate, dihydromorphine, dihydroxyaluminumacetylsalicylate, dimenoxadol, dimepheptanol, dimethylthiambutene,dioxaphetyl butyrate, dipipanone, diprocetyl, dipyrone, ditazol,droxicam, emorfazone, enfenamic acid, enoxolone, epirizole, eptazocine,etersalate, ethenzamide, ethoheptazine, ethoxazene,ethylmethylthiambutene, ethylmorphine, etodolac, etofenamate,etonitazene, eugenol, felbinac, fenbufen, fenclozic acid, fendosal,fenoprofen, fentanyl, fentiazac, fepradinol, feprazone, floctafenine,fluazacort, flucloronide, flufenamic acid, flumethasone, flunisolide,flunixin, flunoxaprofen, fluocinolone acetonide, fluocinonide,fluocinolone acetonide, fluocortin butyl, fluocortolone, fluoresone,fluorometholone, fluperolone, flupirtine, fluprednidene,fluprednisolone, fluproquazone, flurandrenolide, flurbiprofen,fluticasone, formocortal, fosfosal, gentisic acid, glafenine,glucametacin, glycol salicylate, guaiazulene, halcinonide, halobetasol,halometasone, haloprednone, heroin, hydrocodone, hydrocortamate,hydrocortisone, hydrocortisone acetate, hydrocortisone succinate,hydrocortisone hemisuccinate, hydrocortisone 21-lysinate, hydrocortisonecypionate, hydromorphone, hydroxypethidine, ibufenac, ibuprofen,ibuproxam, imidazole salicylate, indomethacin, indoprofen, isofezolac,isoflupredone, isoflupredone acetate, isoladol, isomethadone, isonixin,isoxepac, isoxicam, ketobemidone, ketoprofen, ketorolac,p-lactophenetide, lefetamine, levallorphan, levorphanol,levophenacyl-morphan, lofentanil, lonazolac, lornoxicam, loxoprofen,lysine acetylsalicylate, mazipredone, meclofenamic acid, medrysone,mefenamic acid, meloxicam, meperidine, meprednisone, meptazinol,mesalamine, metazocine, methadone, methotrimeprazine,methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, methylprednisolone suleptanate, metiazinic acid,metofoline, metopon, mofebutazone, mofezolac, mometasone, morazone,morphine, morphine hydrochloride, morphine sulfate, morpholinesalicylate, myrophine, nabumetone, nalbuphine, nalorphine, 1-naphthylsalicylate, naproxen, narceine, nefopam, nicomorphine, nifenazone,niflumic acid, nimesulide, 5′-nitro-2′-propoxyacetanilide,norlevorphanol, normethadone, normorphine, norpipanone, olsalazine,opium, oxaceprol, oxametacine, oxaprozin, oxycodone, oxymorphone,oxyphenbutazone, papaveretum, paramethasone, paranyline, parsalmide,pentazocine, perisoxal, phenacetin, phenadoxone, phenazocine,phenazopyridine hydrochloride, phenocoll, phenoperidine, phenopyrazone,phenomorphan, phenyl acetylsalicylate, phenylbutazone, phenylsalicylate, phenyramidol, piketoprofen, piminodine, pipebuzone,piperylone, pirazolac, piritramide, piroxicam, pirprofen, pranoprofen,prednicarbate, prednisolone, prednisone, prednival, prednylidene,proglumetacin, proheptazine, promedol, propacetamol, properidine,propiram, propoxyphene, propyphenazone, proquazone, protizinic acid,proxazole, ramifenazone, remifentanil, rimazolium metilsulfate,salacetamide, salicin, salicylamide, salicylamide o-acetic acid,salicylic acid, salicylsulfuric acid, salsalate, salverine, simetride,sufentanil, sulfasalazine, sulindac, superoxide dismutase, suprofen,suxibuzone, talniflumate, tenidap, tenoxicam, terofenamate, tetrandrine,thiazolinobutazone, tiaprofenic acid, tiaramide, tilidine, tinoridine,tixocortol, tolfenamic acid, tolmetin, tramadol, triamcinolone,triamcinolone acetonide, tropesin, viminol, xenbucin, ximoprofen,zaltoprofen and zomepirac.

In an exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered witha selective COX-2 inhibitor for treating or preventing inflammation.Exemplary selective COX-2 inhibitors include, for example, deracoxib,parecoxib, celecoxib, valdecoxib, rofecoxib, etoricoxib, lumiracoxib,2-(3,5-difluorophenyl)-3-[4-(methylsulfonyl)phenyl]-2-cyclopenten-1-one,(S)-6,8-dichloro-2-(triflu-oromethyl)-2H-1-benzopyran-3-carboxylic acid,2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methyl-1-butoxy)-5-[4-(methylsulfonyl)phenyl]-3-(2H)-pyridazinone,4-[5-(4-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide,tert-butyl 1benzyl-4-[(4-oxopiperidin-1-yl}sulfonyl]piperidine-4-carboxylate,4-[5-(phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide,salts and prodrugs thereof.

Flushing

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for reducing theincidence or severity of flushing and/or hot flashes which are symptomsof a disorder. For instance, the subject method includes the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein, alone or in combination with other agents, forreducing incidence or severity of flushing and/or hot flashes in cancerpatients. In other embodiments, the method provides for the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce the incidence or severity of flushing and/orhot flashes in menopausal and post-menopausal woman.

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used as a therapy forreducing the incidence or severity of flushing and/or hot flashes whichare side-effects of another drug therapy, e.g., drug-induced flushing.In certain embodiments, a method for treating and/or preventingdrug-induced flushing comprises administering to a patient in needthereof a formulation comprising at least one flushing inducing compoundand at least one sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein. In other embodiments, a method fortreating drug induced flushing comprises separately administering one ormore compounds that induce flushing and one or more sirtuin-modulatingcompounds, e.g., wherein the sirtuin-modulating compound and flushinginducing agent have not been formulated in the same compositions. Whenusing separate formulations, the sirtuin-modulating compound may beadministered (1) at the same as administration of the flushing inducingagent, (2) intermittently with the flushing inducing agent, (3)staggered relative to administration of the flushing inducing agent, (4)prior to administration of the flushing inducing agent, (5) subsequentto administration of the flushing inducing agent, and (6) variouscombination thereof. Exemplary flushing inducing agents include, forexample, niacin, faloxifene, antidepressants, anti-psychotics,chemotherapeutics, calcium channel blockers, and antibiotics.

In one embodiment, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to reduce flushing sideeffects of a vasodilator or an antilipidemic agent (includinganticholesteremic agents and lipotropic agents). In an exemplaryembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be used to reduce flushingassociated with the administration of niacin.

Nicotinic acid, 3-pyridinecarboxylic acid or niacin, is an antilipidemicagent that is marketed under, for example, the trade names Nicolar®,SloNiacin®, Nicobid® and Time Release Niacin®. Nicotinic acid has beenused for many years in the treatment of lipidemic disorders such ashyperlipidemia, hypercholesterolemia and atherosclerosis. This compoundhas long been known to exhibit the beneficial effects of reducing totalcholesterol, low density lipoproteins or “LDL cholesterol,”triglycerides and apolipoprotein a (Lp(a)) in the human body, whileincreasing desirable high density lipoproteins or “HDL cholesterol”.

Typical doses range from about 1 gram to about 3 grams daily. Nicotinicacid is normally administered two to four times per day after meals,depending upon the dosage form selected. Nicotinic acid is currentlycommercially available in two dosage forms. One dosage form is animmediate or rapid release tablet which should be administered three orfour times per day. Immediate release (“IR”) nicotinic acid formulationsgenerally release nearly all of their nicotinic acid within about 30 to60 minutes following ingestion. The other dosage form is a sustainedrelease form which is suitable for administration two to four times perday. In contrast to IR formulations, sustained release (“SR”) nicotinicacid formulations are designed to release significant quantities of drugfor absorption into the blood stream over specific timed intervals inorder to maintain therapeutic levels of nicotinic acid over an extendedperiod such as 12 or 24 hours after ingestion.

As used herein, the term “nicotinic acid” is meant to encompassnicotinic acid or a compound other than nicotinic acid itself which thebody metabolizes into nicotinic acid, thus producing essentially thesame effect as nicotinic acid. Exemplary compounds that produce aneffect similar to that of nicotinic acid include, for example, nicotinylalcohol tartrate, d-glucitol hexanicotinate, aluminum nicotinate,niceritrol and d,1-alpha-tocopheryl nicotinate. Each such compound willbe collectively referred to herein as “nicotinic acid.”

In another embodiment, the invention provides a method for treatingand/or preventing hyperlipidemia with reduced flushing side effects. Themethod comprises the steps of administering to a subject in need thereofa therapeutically effective amount of nicotinic acid and asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein in an amount sufficient to reduce flushing. In anexemplary embodiment, the nicotinic acid and/or sirtuin-modulatingcompound may be administered nocturnally.

In another representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of raloxifene.Raloxifene acts like estrogen in certain places in the body, but is nota hormone. It helps prevent osteoporosis in women who have reachedmenopause. Osteoporosis causes bones to gradually grow thin, fragile,and more likely to break. Evista slows down the loss of bone mass thatoccurs with menopause, lowering the risk of spine fractures due toosteoporosis. A common side effect of raloxifene is hot flashes(sweating and flushing). This can be uncomfortable for women who alreadyhave hot flashes due to menopause.

In another representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of antidepressants oranti-psychotic agent. For instance, sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein can be used inconjunction (administered separately or together) with a serotoninreuptake inhibitor, a 5HT2 receptor antagonist, an anticonvulsant, anorepinephrine reuptake inhibitor, an α-adrenoreceptor antagonist, anNK-3 antagonist, an NK-1 receptor antagonist, a PDE4 inhibitor, anNeuropeptide Y5 Receptor Antagonists, a D4 receptor antagonist, a 5HT1Areceptor antagonist, a 5HT1D receptor antagonist, a CRF antagonist, amonoamine oxidase inhibitor, or a sedative-hypnotic drug.

In certain embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used as part of atreatment with a serotonin reuptake inhibitor (SRI) to reduce flushing.In certain preferred embodiments, the SRI is a selective serotoninreuptake inhibitor (SSRI), such as a fluoxetinoid (fluoxetine,norfluoxetine) or a nefazodonoid (nefazodone, hydroxynefazodone,oxonefazodone). Other exemplary SSRI's include duloxetine, venlafaxine,milnacipran, citalopram, fluvoxamine, paroxetine and sertraline. Thesirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein can also be used as part of a treatment withsedative-hypnotic drug, such as selected from the group consisting of abenzodiazepine (such as alprazolam, chlordiazepoxide, clonazepam,chlorazepate, clobazam, diazepam, halazepam, lorazepam, oxazepam andprazepam), zolpidem, and barbiturates. In still other embodiments, asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein may be used as part of a treatment with a 5-HT1Areceptor partial agonist, such as selected from the group consisting ofbuspirone, flesinoxan, gepirone and ipsapirone. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteincan also used as part of a treatment with a norepinephrine reuptakeinhibitor, such as selected from tertiary amine tricyclics and secondaryamine tricyclics. Exemplary tertiary amine tricyclic includeamitriptyline, clomipramine, doxepin, imipramine and trimipramine.Exemplary secondary amine tricyclic include amoxapine, desipramine,maprotiline, nortriptyline and protriptyline. In certain embodiments,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used as part of a treatment with a monoamineoxidase inhibitor, such as selected from the group consisting ofisocarboxazid, phenelzine, tranylcypromine, selegiline and moclobemide.

In still another representative embodiment, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedto reduce flushing side effects of chemotherapeutic agents, such ascyclophosphamide, tamoxifen.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of calcium channel blockers, such as amlodipine.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of antibiotics. For example, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteincan be used in combination with levofloxacin. Levofloxacin is used totreat infections of the sinuses, skin, lungs, ears, airways, bones, andjoints caused by susceptible bacteria. Levofloxacin also is frequentlyused to treat urinary infections, including those resistant to otherantibiotics, as well as prostatitis. Levofloxacin is effective intreating infectious diarrheas caused by E. coli, campylobacter jejuni,and shigella bacteria. Levofloxacin also can be used to treat variousobstetric infections, including mastitis.

Ocular Disorders

One aspect of the present invention is a method for inhibiting, reducingor otherwise treating vision impairment by administering to a patient atherapeutic dosage of sirtuin modulator selected from a compounddisclosed herein, or a pharmaceutically acceptable salt, prodrug or ametabolic derivative thereof.

In certain aspects of the invention, the vision impairment is caused bydamage to the optic nerve or central nervous system. In particularembodiments, optic nerve damage is caused by high intraocular pressure,such as that created by glaucoma. In other particular embodiments, opticnerve damage is caused by swelling of the nerve, which is oftenassociated with an infection or an immune (e.g., autoimmune) responsesuch as in optic neuritis.

Glaucoma describes a group of disorders which are associated with avisual field defect, cupping of the optic disc, and optic nerve damage.These are commonly referred to as glaucomatous optic neuropathies. Mostglaucomas are usually, but not always, associated with a rise inintraocular pressure. Exemplary forms of glaucoma include Glaucoma andPenetrating Keratoplasty, Acute Angle Closure, Chronic Angle Closure,Chronic Open Angle, Angle Recession, Aphakic and Pseudophakic,Drug-Induced, Hyphema, Intraocular Tumors, Juvenile, Lens-Particle, LowTension, Malignant, Neovascular, Phacolytic, Phacomorphic, Pigmentary,Plateau Iris, Primary Congenital, Primary Open Angle, Pseudoexfoliation,Secondary Congenital, Adult Suspect, Unilateral, Uveitic, OcularHypertension, Ocular Hypotony, Posner-Schlossman Syndrome and ScleralExpansion Procedure in Ocular Hypertension & Primary Open-angleGlaucoma.

Intraocular pressure can also be increased by various surgicalprocedures, such as phacoemulsification (i.e., cataract surgery) andimplantation of structures such as an artificial lens. In addition,spinal surgeries in particular, or any surgery in which the patient isprone for an extended period of time can lead to increased interocularpressure.

Optic neuritis (ON) is inflammation of the optic nerve and causes acuteloss of vision. It is highly associated with multiple sclerosis (MS) as15-25% of MS patients initially present with ON, and 50-75% of ONpatients are diagnosed with MS. ON is also associated with infection(e.g., viral infection, meningitis, syphilis), inflammation (e.g., froma vaccine), infiltration and ischemia.

Another condition leading to optic nerve damage is anterior ischemicoptic neuropathy (AION). There are two types of AION. Arteritic AION isdue to giant cell arteritis (vasculitis) and leads to acute vision loss.Non-arteritic AION encompasses all cases of ischemic optic neuropathyother than those due to giant cell arteritis. The pathophysiology ofAION is unclear although it appears to incorporate both inflammatory andischemic mechanisms.

Other damage to the optic nerve is typically associated withdemyleination, inflammation, ischemia, toxins, or trauma to the opticnerve. Exemplary conditions where the optic nerve is damaged includeDemyelinating Optic Neuropathy (Optic Neuritis, Retrobulbar OpticNeuritis), Optic Nerve Sheath Meningioma, Adult Optic Neuritis,Childhood Optic Neuritis, Anterior Ischemic Optic Neuropathy, PosteriorIschemic Optic Neuropathy, Compressive Optic Neuropathy, Papilledema,Pseudopapilledema and Toxic/Nutritional Optic Neuropathy.

Other neurological conditions associated with vision loss, albeit notdirectly associated with damage to the optic nerve, include Amblyopia,Bells Palsy, Chronic Progressive External Ophthalmoplegia, MultipleSclerosis, Pseudotumor Cerebri and Trigeminal Neuralgia.

In certain aspects of the invention, the vision impairment is caused byretinal damage. In particular embodiments, retinal damage is caused bydisturbances in blood flow to the eye (e.g., arteriosclerosis,vasculitis). In particular embodiments, retinal damage is caused bydisruption of the macula (e.g., exudative or non-exudative maculardegeneration).

Exemplary retinal diseases include Exudative Age Related MacularDegeneration, Nonexudative Age Related Macular Degeneration, RetinalElectronic Prosthesis and RPE Transplantation Age Related MacularDegeneration, Acute Multifocal Placoid Pigment Epitheliopathy, AcuteRetinal Necrosis, Best Disease, Branch Retinal Artery Occlusion, BranchRetinal Vein Occlusion, Cancer Associated and Related AutoimmuneRetinopathies, Central Retinal Artery Occlusion, Central Retinal VeinOcclusion, Central Serous Chorioretinopathy, Eales Disease, EpimacularMembrane, Lattice Degeneration, Macroaneurysm, Diabetic Macular Edema,Irvine-Gass Macular Edema, Macular Hole, Subretinal NeovascularMembranes, Diffuse Unilateral Subacute Neuroretinitis, NonpseudophakicCystoid Macular Edema, Presumed Ocular Histoplasmosis Syndrome,Exudative Retinal Detachment, Postoperative Retinal Detachment,Proliferative Retinal Detachment, Rhegmatogenous Retinal Detachment,Tractional Retinal Detachment, Retinitis Pigmentosa, CMV Retinitis,Retinoblastoma, Retinopathy of Prematurity, Birdshot Retinopathy,Background Diabetic Retinopathy, Proliferative Diabetic Retinopathy,Hemoglobinopathies Retinopathy, Purtscher Retinopathy, ValsalvaRetinopathy, Juvenile Retinoschisis, Senile Retinoschisis, TersonSyndrome and White Dot Syndromes.

Other exemplary diseases include ocular bacterial infections (e.g.conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viralinfections (e.g. Ocular Herpes Simplex Virus, Varicella Zoster Virus,Cytomegalovirus retinitis, Human Immunodeficiency Virus (HIV)) as wellas progressive outer retinal necrosis secondary to HIV or otherHIV-associated and other immunodeficiency-associated ocular diseases. Inaddition, ocular diseases include fungal infections (e.g. Candidachoroiditis, histoplasmosis), protozoal infections (e.g. toxoplasmosis)and others such as ocular toxocariasis and sarcoidosis.

One aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing treatment with achemotherapeutic drug (e.g., a neurotoxic drug, a drug that raisesintraocular pressure such as a steroid), by administering to the subjectin need of such treatment a therapeutic dosage of a sirtuin modulatordisclosed herein.

Another aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing surgery, includingocular or other surgeries performed in the prone position such as spinalcord surgery, by administering to the subject in need of such treatmenta therapeutic dosage of a sirtuin modulator disclosed herein. Ocularsurgeries include cataract, iridotomy and lens replacements.

Another aspect of the invention is the treatment, including inhibitionand prophylactic treatment, of age related ocular diseases includecataracts, dry eye, retinal damage and the like, by administering to thesubject in need of such treatment a therapeutic dosage of a sirtuinmodulator disclosed herein.

The formation of cataracts is associated with several biochemicalchanges in the lens of the eye, such as decreased levels of antioxidantsascorbic acid and glutathione, increased lipid, amino acid and proteinoxidation, increased sodium and calcium, loss of amino acids anddecreased lens metabolism. The lens, which lacks blood vessels, issuspended in extracellular fluids in the anterior part of the eye.Nutrients, such as ascorbic acid, glutathione, vitamin E, selenium,bioflavonoids and carotenoids are required to maintain the transparencyof the lens. Low levels of selenium results in an increase of freeradical-inducing hydrogen peroxide, which is neutralized by theselenium-dependent antioxidant enzyme glutathione peroxidase.Lens-protective glutathione peroxidase is also dependent on the aminoacids methionine, cysteine, glycine and glutamic acid.

Cataracts can also develop due to an inability to properly metabolizegalactose found in dairy products that contain lactose, a disaccharidecomposed of the monosaccharide galactose and glucose. Cataracts can beprevented, delayed, slowed and possibly even reversed if detected earlyand metabolically corrected.

Retinal damage is attributed, inter alia, to free radical initiatedreactions in glaucoma, diabetic retinopathy and age-related maculardegeneration (AMD). The eye is a part of the central nervous system andhas limited regenerative capability. The retina is composed of numerousnerve cells which contain the highest concentration of polyunsaturatedfatty acids (PFA) and subject to oxidation. Free radicals are generatedby UV light entering the eye and mitochondria in the rods and cones,which generate the energy necessary to transform light into visualimpulses. Free radicals cause peroxidation of the PFA by hydroxyl orsuperoxide radicals which in turn propagate additional free radicals.The free radicals cause temporary or permanent damage to retinal tissue.

Glaucoma is usually viewed as a disorder that causes an elevatedintraocular pressure (IOP) that results in permanent damage to theretinal nerve fibers, but a sixth of all glaucoma cases do not developan elevated IOP. This disorder is now perceived as one of reducedvascular perfusion and an increase in neurotoxic factors. Recent studieshave implicated elevated levels of glutamate, nitric oxide andperoxynitirite in the eye as the causes of the death of retinal ganglioncells. Neuroprotective agents may be the future of glaucoma care. Forexample, nitric oxide synthase inhibitors block the formation ofperoxynitrite from nitric oxide and superoxide. In a recent study,animals treated with aminoguanidine, a nitric oxide synthase inhibitor,had a reduction in the loss of retinal ganglion cells. It was concludedthat nitric oxide in the eye caused cytotoxicity in many tissues andneurotoxicity in the central nervous system.

Diabetic retinopathy occurs when the underlying blood vessels developmicrovascular abnormalities consisting primarily of microaneurysms andintraretinal hemorrhages. Oxidative metabolites are directly involvedwith the pathogenesis of diabetic retinopathy and free radicals augmentthe generation of growth factors that lead to enhanced proliferativeactivity. Nitric oxide produced by endothelial cells of the vessels mayalso cause smooth muscle cells to relax and result in vasodilation ofsegments of the vessel. Ischemia and hypoxia of the retina occur afterthickening of the arterial basement membrane, endothelial proliferationand loss of pericytes. The inadequate oxygenation causes capillaryobliteration or nonperfusion, arteriolar-venular shunts, sluggish bloodflow and an impaired ability of RBCs to release oxygen. Lipidperoxidation of the retinal tissues also occurs as a result of freeradical damage.

The macula is responsible for our acute central vision and composed oflight-sensing cells (cones) while the underlying retinal pigmentepithelium (RPE) and choroid nourish and help remove waste materials.The RPE nourishes the cones with the vitamin A substrate for thephotosensitive pigments and digests the cones shed outer tips. RPE isexposed to high levels of UV radiation, and secretes factors thatinhibit angiogenesis. The choroid contains a dense vascular network thatprovides nutrients and removes the waste materials.

In AMD, the shed cone tips become indigestible by the RPE, where thecells swell and die after collecting too much undigested material.Collections of undigested waste material, called drusen, form under theRPE. Photoxic damage also causes the accumulation of lipofuscin in RPEcells. The intracellular lipofuscin and accumulation of drusen inBruch's membrane interferes with the transport of oxygen and nutrientsto the retinal tissues, and ultimately leads to RPE and photoreceptordysfunction. In exudative AMD, blood vessels grow from thechoriocapillaris through defects in Bruch's membrane and may grow underthe RPE, detaching it from the choroid, and leaking fluid or bleeding.

Macular pigment, one of the protective factors that prevent sunlightfrom damaging the retina, is formed by the accumulation of nutritionallyderived carotenoids, such as lutein, the fatty yellow pigment thatserves as a delivery vehicle for other important nutrients andzeaxanthin. Antioxidants such as vitamins C and E, beta-carotene andlutein, as well as zinc, selenium and copper, are all found in thehealthy macula. In addition to providing nourishment, these antioxidantsprotect against free radical damage that initiates macular degeneration.

Another aspect of the invention is the prevention or treatment of damageto the eye caused by stress, chemical insult or radiation, byadministering to the subject in need of such treatment a therapeuticdosage of a sirtuin modulator disclosed herein. Radiation orelectromagnetic damage to the eye can include that caused by CRT's orexposure to sunlight or UV.

In one embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of ocular disorders orsecondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more therapeutic agents for the treatment of an ocular disorder.For example, one or more sirtuin-activating compounds can be combinedwith an effective amount of one or more of: an agent that reducesintraocular pressure, an agent for treating glaucoma, an agent fortreating optic neuritis, an agent for treating CMV Retinopathy, an agentfor treating multiple sclerosis, and/or an antibiotic, etc.

In one embodiment, a sirtuin modulator can be administered inconjunction with a therapy for reducing intraocular pressure. One groupof therapies involves blocking aqueous production. For example, topicalbeta-adrenergic antagonists (timolol and betaxolol) decrease aqueousproduction. Topical timolol causes IOP to fall in 30 minutes with peakeffects in 1-2 hours. A reasonable regimen is Timoptic 0.5%, one dropevery 30 minutes for 2 doses. The carbonic anhydrase inhibitor,acetazolamide, also decreases aqueous production and should be given inconjunction with topical beta-antagonists. An initial dose of 500 mg isadministered followed by 250 mg every 6 hours. This medication may begiven orally, intramuscularly, or intravenously. In addition, alpha2-agonists (e.g., Apraclonidine) act by decreasing aqueous production.Their effects are additive to topically administered beta-blockers. Theyhave been approved for use in controlling an acute rise in pressurefollowing anterior chamber laser procedures, but has been reportedeffective in treating acute closed-angle glaucoma. A reasonable regimenis 1 drop every 30 minutes for 2 doses.

A second group of therapies for reducing intraocular pressure involvereducing vitreous volume. Hyperosmotic agents can be used to treat anacute attack. These agents draw water out of the globe by making theblood hyperosmolar. Oral glycerol in a dose of 1 mL/g in a cold 50%solution (mixed with lemon juice to make it more palatable) often isused. Glycerol is converted to glucose in the liver; persons withdiabetes may need additional insulin if they become hyperglycemic afterreceiving glycerol. Oral isosorbide is a metabolically inert alcoholthat also can be used as an osmotic agent for patients with acuteangle-closure glaucoma. Usual dose is 100 g taken p o. (220 cc of a 45%solution). This inert alcohol should not be confused with isosorbidedinitrate, a nitrate-based cardiac medication used for angina and forcongestive heart failure. Intravenous mannitol in a dose of 1.0-1.5mg/kg also is effective and is well tolerated in patients with nauseaand vomiting. These hyperosmotic agents should be used with caution inany patient with a history of congestive heart failure.

A third group of therapies involve facilitating aqueous outflow from theeye. Miotic agents pull the iris from the iridocorneal angle and mayhelp to relieve the obstruction of the trabecular meshwork by theperipheral iris. Pilocarpine 2% (blue eyes)-4% (brown eyes) can beadministered every 15 minutes for the first 1-2 hours. More frequentadministration or higher doses may precipitate a systemic cholinergiccrisis. NSAIDS are sometimes used to reduce inflammation.

Exemplary therapeutic agents for reducing intraocular pressure includeALPHAGAN® P (Allergan) (brimonidine tartrate ophthalmic solution),AZOPT® (Alcon) (brinzolamide ophthalmic suspension), BETAGAN® (Allergan)(levobunolol hydrochloride ophthalmic solution, USP), BETIMOL®(Vistakon) (timolol ophthalmic solution), BETOPTIC S® (Alcon) (betaxololHCl), BRIMONIDINE TARTRATE (Bausch & Lomb), CARTEOLOL HYDROCHLORIDE(Bausch & Lomb), COSOPT® (Merck) (dorzolamide hydrochloride-timololmaleate ophthalmic solution), LUMIGAN® (Allergan) (bimatoprostophthalmic solution), OPTIPRANOLOL® (Bausch & Lomb) (metipranololophthalmic solution), TIMOLOL GFS (Falcon) (timolol maleate ophthalmicgel forming solution), TIMOPTIC® (Merck) (timolol maleate ophthalmicsolution), TRAVATAN® (Alcon) (travoprost ophthalmic solution), TRUSOPT®(Merck) (dorzolamide hydrochloride ophthalmic solution) and XALATAN®(Pharmacia & Upjohn) (latanoprost ophthalmic solution).

In one embodiment, a sirtuin modulator can be administered inconjunction with a therapy for treating and/or preventing glaucoma. Anexample of a glaucoma drug is DARANIDE® Tablets (Merck)(Dichlorphenamide).

In one embodiment, a sirtuin modulator can be administered inconjunction with a therapy for treating and/or preventing opticneuritis. Examples of drugs for optic neuritis include DECADRON®Phosphate Injection (Merck) (Dexamethasone Sodium Phosphate),DEPO-MEDROL® (Pharmacia & Upjohn)(methylprednisolone acetate),HYDROCORTONE® Tablets (Merck) (Hydrocortisone), ORAPRED® (Biomarin)(prednisolone sodium phosphate oral solution) and PEDIAPRED® (Celltech)(prednisolone sodium phosphate, USP).

In one embodiment, a sirtuin modulator can be administered inconjunction with a therapy for treating and/or preventing CMVRetinopathy. Treatments for CMV retinopathy include CYTOVENE®(ganciclovir capsules) and VALCYTE® (Roche Laboratories) (valganciclovirhydrochloride tablets).

In one embodiment, a sirtuin modulator can be administered inconjunction with a therapy for treating and/or preventing multiplesclerosis. Examples of such drugs include DANTRIUM® (Procter & GamblePharmaceuticals) (dantrolene sodium), NOVANTRONE® (Serono)(mitoxantrone), AVONEX® (Biogen Idec) (Interferon beta-1a), BETASERON®(Berlex) (Interferon beta-1b), COPAXONE® (Teva Neuroscience) (glatirameracetate injection) and REBIF® (Pfizer) (interferon beta-1a).

In addition, macrolide and/or mycophenolic acid, which has multipleactivities, can be co-administered with a sirtuin modulator. Macrolideantibiotics include tacrolimus, cyclosporine, sirolimus, everolimus,ascomycin, erythromycin, azithromycin, clarithromycin, clindamycin,lincomycin, dirithromycin, josamycin, spiramycin, diacetyl-midecamycin,tylosin, roxithromycin, ABT-773, telithromycin, leucomycins, andlincosamide.

Mitochondrial-Associated Diseases and Disorders

In certain embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity. The methods involve administering to a subject in need thereofa therapeutically effective amount of a sirtuin activating compound.Increased mitochondrial activity refers to increasing activity of themitochondria while maintaining the overall numbers of mitochondria(e.g., mitochondrial mass), increasing the numbers of mitochondriathereby increasing mitochondrial activity (e.g., by stimulatingmitochondrial biogenesis), or combinations thereof. In certainembodiments, diseases and disorders that would benefit from increasedmitochondrial activity include diseases or disorders associated withmitochondrial dysfunction.

In certain embodiments, methods for treating diseases or disorders thatwould benefit from increased mitochondrial activity may compriseidentifying a subject suffering from a mitochondrial dysfunction.Methods for diagnosing a mitochondrial dysfunction may involve moleculargenetic, pathologic and/or biochemical analysis are summarized in Cohenand Gold, Cleveland Clinic Journal of Medicine, 68: 625-642 (2001). Onemethod for diagnosing a mitochondrial dysfunction is the Thor-Byrne-ierscale (see e.g., Cohen and Gold, supra; Collin S. et al., Eur Neurol.36: 260-267 (1996)). Other methods for determining mitochondrial numberand function include, for example, enzymatic assays (e.g., amitochondrial enzyme or an ATP biosynthesis factor such as an ETC enzymeor a Krebs cycle enzyme), determination or mitochondrial mass,mitochondrial volume, and/or mitochondrial number, quantification ofmitochondrial DNA, monitoring intracellular calcium homeostasis and/orcellular responses to perturbations of this homeostasis, evaluation ofresponse to an apoptogenic stimulus, determination of free radicalproduction. Such methods are known in the art and are described, forexample, in U.S. Patent Publication No. 2002/0049176 and referencescited therein.

Mitochondria are critical for the survival and proper function of almostall types of eukaryotic cells. Mitochondria in virtually any cell typecan have congenital or acquired defects that affect their function.Thus, the clinically significant signs and symptoms of mitochondrialdefects affecting respiratory chain function are heterogeneous andvariable depending on the distribution of defective mitochondria amongcells and the severity of their deficits, and upon physiological demandsupon the affected cells. Nondividing tissues with high energyrequirements, e.g. nervous tissue, skeletal muscle and cardiac muscleare particularly susceptible to mitochondrial respiratory chaindysfunction, but any organ system can be affected.

Diseases and disorders associated with mitochondrial dysfunction includediseases and disorders in which deficits in mitochondrial respiratorychain activity contribute to the development of pathophysiology of suchdiseases or disorders in a mammal. This includes 1) congenital geneticdeficiencies in activity of one or more components of the mitochondrialrespiratory chain; and 2) acquired deficiencies in the activity of oneor more components of the mitochondrial respiratory chain, wherein suchdeficiencies are caused by a) oxidative damage during aging; b) elevatedintracellular calcium; c) exposure of affected cells to nitric oxide; d)hypoxia or ischemia; e) microtubule-associated deficits in axonaltransport of mitochondria, or f) expression of mitochondrial uncouplingproteins.

Diseases or disorders that would benefit from increased mitochondrialactivity generally include for example, diseases in which free radicalmediated oxidative injury leads to tissue degeneration, diseases inwhich cells inappropriately undergo apoptosis, and diseases in whichcells fail to undergo apoptosis. Exemplary diseases or disorders thatwould benefit from increased mitochondrial activity include, forexample, AD (Alzheimer's Disease), ADPD (Alzheimer's Disease andParkinsons's Disease), AMDF (Ataxia, Myoclonus and Deafness),auto-immune disease, cancer, CIPO (Chronic Intestinal Pseudoobstructionwith myopathy and Opthalmoplegia), congenital muscular dystrophy, CPEO(Chronic Progressive External Opthalmoplegia), DEAF (Maternallyinherited DEAFness or aminoglycoside-induced DEAFness), DEMCHO (Dementiaand Chorea), diabetes mellitis (Type I or Type II), DIDMOAD (DiabetesInsipidus, Diabetes Mellitus, Optic Atrophy, Deafness), DMDF (DiabetesMellitus and Deafness), dystonia, Exercise Intolerance, ESOC (Epilepsy,Strokes, Optic atrophy, and Cognitive decline), FBSN (Familial BilateralStriatal Necrosis), FICP (Fatal Infantile Cardiomyopathy Plus, aMELAS-associated cardiomyopathy), GER (Gastrointestinal Reflux), HD(Huntington's Disease), KSS (Kearns Sayre Syndrome), “later-onset”myopathy, LDYT (Leber's hereditary optic neuropathy and DYsTonia),Leigh's Syndrome, LHON (Leber Hereditary Optic Neuropathy), LIMM (LethalInfantile Mitochondrial Myopathy), MDM (Myopathy and Diabetes Mellitus),MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-likeepisodes), MEPR (Myoclonic Epilepsy and Psychomotor Regression), MERME(MERRF/MELAS overlap disease), MERRF (Myoclonic Epilepsy and Ragged RedMuscle Fibers), MHCM (Maternally Inherited Hypertrophic CardioMyopathy),MICM (Maternally Inherited Cardiomyopathy), MILS (Maternally InheritedLeigh Syndrome), Mitochondrial Encephalocardiomyopathy, MitochondrialEncephalomyopathy, MM (Mitochondrial Myopathy), MMC (Maternal Myopathyand Cardiomyopathy), MNGIE (Myopathy and external ophthalmoplegia,Neuropathy, Gastro-Intestinal, Encephalopathy), MultisystemMitochondrial Disorder (myopathy, encephalopathy, blindness, hearingloss, peripheral neuropathy), NARP (Neurogenic muscle weakness, Ataxia,and Retinitis Pigmentosa; alternate phenotype at this locus is reportedas Leigh Disease), PD (Parkinson's Disease), Pearson's Syndrome, PEM(Progressive Encephalopathy), PEO (Progressive External Opthalmoplegia),PME (Progressive Myoclonus Epilepsy), PMPS (Pearson Marrow-PancreasSyndrome), psoriasis, RTT (Rett Syndrome), schizophrenia, SIDS (SuddenInfant Death Syndrome), SNHL (Sensorineural Hearing Loss), VariedFamilial Presentation (clinical manifestations range from spasticparaparesis to multisystem progressive disorder & fatal cardiomyopathyto truncal ataxia, dysarthria, severe hearing loss, mental regression,ptosis, ophthalmoparesis, distal cyclones, and diabetes mellitus), orWolfram syndrome.

Other diseases and disorders that would benefit from increasedmitochondrial activity include, for example, Friedreich's ataxia andother ataxias, amyotrophic lateral sclerosis (ALS) and other motorneuron diseases, macular degeneration, epilepsy, Alpers syndrome,Multiple mitochondrial DNA deletion syndrome, MTDNA depletion syndrome,Complex I deficiency, Complex II (SDH) deficiency, Complex IIIdeficiency, Cytochrome c oxidase (COX, Complex IV) deficiency, Complex Vdeficiency, Adenine Nucleotide Translocator (ANT) deficiency, Pyruvatedehydrogenase (PDH) deficiency, Ethylmalonic aciduria with lacticacidemia, 3-Methyl glutaconic aciduria with lactic acidemia, Refractoryepilepsy with declines during infection, Asperger syndrome with declinesduring infection, Autism with declines during infection, Attentiondeficit hyperactivity disorder (ADHD), Cerebral palsy with declinesduring infection, Dyslexia with declines during infection, materiallyinherited thrombocytopenia and leukemia syndrome, MARIAHS syndrome(Mitrochondrial ataxia, recurrent infections, aphasia,hypouricemia/hypomyelination, seizures, and dicarboxylic aciduria), ND6dystonia, Cyclic vomiting syndrome with declines during infection,3-Hydroxy isobutryic aciduria with lactic acidemia, Diabetes mellituswith lactic acidemia, Uridine responsive neurologic syndrome (URNS),Dilated cardiomyopathy, Splenic Lymphoma, and Renal TubularAcidosis/Diabetes/Ataxis syndrome.

In other embodiments, the invention provides methods for treating asubject suffering from mitochondrial disorders arising from, but notlimited to, post-traumatic head injury and cerebral edema, stroke(invention methods useful for preventing or preventing reperfusioninjury), Lewy body dementia, hepatorenal syndrome, acute liver failure,NASH (non-alcoholic steatohepatitis), Anti-metastasis/prodifferentiationtherapy of cancer, idiopathic congestive heart failure, atrialfibrilation (non-valvular), Wolff-Parkinson-White Syndrome, idiopathicheart block, prevention of reperfusion injury in acute myocardialinfarctions, familial migraines, irritable bowel syndrome, secondaryprevention of non-Q wave myocardial infarctions, Premenstrual syndrome,Prevention of renal failure in hepatorenal syndrome, anti-phospholipidantibody syndrome, eclampsia/pre-eclampsia, oopause infertility,ischemic heart disease/angina, and Shy-Drager and unclassifieddysautonomia syndromes.

In still another embodiment, there are provided methods for thetreatment of mitochondrial disorders associated with pharmacologicaldrug-related side effects. Types of pharmaceutical agents that areassociated with mitochondrial disorders include reverse transcriptaseinhibitors, protease inhibitors, inhibitors of DHOD, and the like.Examples of reverse transcriptase inhibitors include, for example,Azidothymidine (AZT), Stavudine (D4T), Zalcitabine (ddC), Didanosine(DDI), Fluoroiodoarauracil (FIAU), Lamivudine (3TC), Abacavir and thelike. Examples of protease inhibitors include, for example, Ritonavir,Indinavir, Saquinavir, Nelfinavir and the like. Examples of inhibitorsof dihydroorotate dehydrogenase (DHOD) include, for example,Leflunomide, Brequinar, and the like.

Reverse transcriptase inhibitors not only inhibit reverse transcriptasebut also polymerase gamma which is required for mitochondrial function.Inhibition of polymerase gamma activity (e.g., with a reversetranscriptase inhibitor) therefore leads to mitochondrial dysfunctionand/or a reduced mitochondrial mass which manifests itself in patientsas hyperlactatemia. This type of condition may benefit from an increasein the number of mitochondria and/or an improvement in mitochondrialfunction, e.g., by administration of a sirtuin activating compound.

Common symptoms of mitochondrial diseases include cardiomyopathy, muscleweakness and atrophy, developmental delays (involving motor, language,cognitive or executive function), ataxia, epilepsy, renal tubularacidosis, peripheral neuropathy, optic neuropathy, autonomic neuropathy,neurogenic bowel dysfunction, sensorineural deafness, neurogenic bladderdysfunction, dilating cardiomyopathy, migraine, hepatic failure, lacticacidemia, and diabetes mellitus.

In certain embodiments, the invention provides methods for treating adisease or disorder that would benefit from increased mitochondrialactivity that involves administering to a subject in need thereof one ormore sirtuin activating compounds in combination with anothertherapeutic agent such as, for example, an agent useful for treatingmitochondrial dysfunction (such as antioxidants, vitamins, orrespiratory chain cofactors), an agent useful for reducing a symptomassociated with a disease or disorder involving mitochondrialdysfunction (such as, an anti-seizure agent, an agent useful foralleviating neuropathic pain, an agent for treating cardiacdysfunction), a cardiovascular agent (as described further below), achemotherapeutic agent (as described further below), or ananti-neurodegeneration agent (as described further below). In anexemplary embodiment, the invention provides methods for treating adisease or disorder that would benefit from increased mitochondrialactivity that involves administering to a subject in need thereof one ormore sirtuin activating compounds in combination with one or more of thefollowing: coenzyme Q₁₀, L-carnitine, thiamine, riboflavin, niacinamide,folate, vitamin E, selenium, lipoic acid, or prednisone. Compositionscomprising such combinations are also provided herein.

In exemplary embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity by administering to a subject a therapeutically effectiveamount of a sirtuin activating compound. Exemplary diseases or disordersinclude, for example, neuromuscular disorders (e.g., Friedreich'sAtaxia, muscular dystrophy, multiple sclerosis, etc.), disorders ofneuronal instability (e.g., seizure disorders, migraine, etc.),developmental delay, neurodegenerative disorders (e.g., Alzheimer'sDisease, Parkinson's Disease, amyotrophic lateral sclerosis, etc.),ischemia, renal tubular acidosis, age-related neurodegeneration andcognitive decline, chemotherapy fatigue, age-related orchemotherapy-induced menopause or irregularities of menstrual cycling orovulation, mitochondrial myopathies, mitochondrial damage (e.g., calciumaccumulation, excitotoxicity, nitric oxide exposure, hypoxia, etc.), andmitochondrial deregulation.

A gene defect underlying Friedreich's Ataxia (FA), the most commonhereditary ataxia, was recently identified and is designated “frataxin”.In FA, after a period of normal development, deficits in coordinationdevelop which progress to paralysis and death, typically between theages of 30 and 40. The tissues affected most severely are the spinalcord, peripheral nerves, myocardium, and pancreas. Patients typicallylose motor control and are confined to wheel chairs, and are commonlyafflicted with heart failure and diabetes. The genetic basis for FAinvolves GAA trinucleotide repeats in an intron region of the geneencoding frataxin. The presence of these repeats results in reducedtranscription and expression of the gene. Frataxin is involved inregulation of mitochondrial iron content. When cellular frataxin contentis subnormal, excess iron accumulates in mitochondria, promotingoxidative damage and consequent mitochondrial degeneration anddysfunction. When intermediate numbers of GAA repeats are present in thefrataxin gene intron, the severe clinical phenotype of ataxia may notdevelop. However, these intermediate-length trinucleotide extensions arefound in 25 to 30% of patients with non-insulin dependent diabetesmellitus, compared to about 5% of the nondiabetic population. In certainembodiments, sirtuin activating compounds may be used for treatingpatients with disorders related to deficiencies or defects in frataxin,including Friedreich's Ataxia, myocardial dysfunction, diabetes mellitusand complications of diabetes like peripheral neuropathy.

Muscular dystrophy refers to a family of diseases involvingdeterioration of neuromuscular structure and function, often resultingin atrophy of skeletal muscle and myocardial dysfunction. In the case ofDuchenne muscular dystrophy, mutations or deficits in a specificprotein, dystrophin, are implicated in its etiology. Mice with theirdystrophin genes inactivated display some characteristics of musculardystrophy, and have an approximately 50% deficit in mitochondrialrespiratory chain activity. A final common pathway for neuromusculardegeneration in most cases is calcium-mediated impairment ofmitochondrial function. In certain embodiments, sirtuin activatingcompounds may be used for reducing the rate of decline in muscularfunctional capacities and for improving muscular functional status inpatients with muscular dystrophy.

Multiple sclerosis (MS) is a neuromuscular disease characterized byfocal inflammatory and autoimmune degeneration of cerebral white matter.Periodic exacerbations or attacks are significantly correlated withupper respiratory tract and other infections, both bacterial and viral,indicating that mitochondrial dysfunction plays a role in MS. Depressionof neuronal mitochondrial respiratory chain activity caused by NitricOxide (produced by astrocytes and other cells involved in inflammation)is implicated as a molecular mechanism contributing to MS. In certainembodiments, sirtuin activating compounds may be used for treatment ofpatients with multiple sclerosis, both prophylactically and duringepisodes of disease exacerbation.

Epilepsy is often present in patients with mitochondrial cytopathies,involving a range of seizure severity and frequency, e.g. absence,tonic, atonic, myoclonic, and status epilepticus, occurring in isolatedepisodes or many times daily. In certain embodiments, sirtuin activatingcompounds may be used for treating patients with seizures secondary tomitochondrial dysfunction, including reducing frequency and severity ofseizure activity.

Metabolic studies on patients with recurrent migraine headaches indicatethat deficits in mitochondrial activity are commonly associated withthis disorder, manifesting as impaired-oxidative phosphorylation andexcess lactate production. Such deficits are not necessarily due togenetic defects in mitochondrial DNA. Migraineurs are hypersensitive tonitric oxide, an endogenous inhibitor of Cytochrome c Oxidase. Inaddition, patients with mitochondrial cytopathies, e.g. MELAS, oftenhave recurrent migraines. In certain embodiments, sirtuin activatingcompounds may be used for treating patients with recurrent migraineheadaches, including headaches refractory to ergot compounds orserotonin receptor antagonists.

Delays in neurological or neuropsychological development are often foundin children with mitochondrial diseases. Development and remodeling ofneural connections requires intensive biosynthetic activity,particularly involving synthesis of neuronal membranes and myelin, bothof which require pyrimidine nucleotides as cofactors. Uridinenucleotides are involved inactivation and transfer of sugars toglycolipids and glycoproteins. Cytidine nucleotides are derived fromuridine nucleotides, and are crucial for synthesis of major membranephospholipid constituents like phosphatidylcholine, which receives itscholine moiety from cytidine diphosphocholine. In the case ofmitochondrial dysfunction (due to either mitochondrial DNA defects orany of the acquired or conditional deficits like exicitoxic or nitricoxide-mediated mitochondrial dysfunction) or other conditions resultingin impaired pyrimidine synthesis, cell proliferation and axonalextension is impaired at crucial stages in development of neuronalinterconnections and circuits, resulting in delayed or arresteddevelopment of neuropsychological functions like language, motor,social, executive function, and cognitive skills. In autism for example,magnetic resonance spectroscopy measurements of cerebral phosphatecompounds indicates that there is global undersynthesis of membranes andmembrane precursors indicated by reduced levels of uridinediphospho-sugars, and cytidine nucleotide derivatives involved inmembrane synthesis. Disorders characterized by developmental delayinclude Rett's Syndrome, pervasive developmental delay (or PDD-NOS“pervasive developmental delay not otherwise specified” to distinguishit from specific subcategories like autism), autism, Asperger'sSyndrome, and Attention Deficit/Hyperactivity Disorder (ADHD), which isbecoming recognized as a delay or lag in development of neural circuitryunderlying executive functions. In certain embodiments, sirtuinactivating compounds may be useful for treating patients withneurodevelopmental delays (e.g., involving motor, language, executivefunction, and cognitive skills), or other delays or arrests ofneurological and neuropsychological development in the nervous systemand somatic development in non-neural tissues like muscle and endocrineglands.

The two most significant severe neurodegenerative diseases associatedwith aging, Alzheimer's Disease (AD) and Parkinson's Disease (PD), bothinvolve mitochondrial dysfunction in their pathogenesis. Complex Ideficiencies in particular are frequently found not only in thenigrostriatal neurons that degenerate in Parkinson's disease, but alsoin peripheral tissues and cells like muscle and platelets of Parkinson'sDisease patients. In Alzheimer's Disease, mitochondrial respiratorychain activity is often depressed, especially Complex IV (Cytochrome cOxidase). Moreover, mitochondrial respiratory function altogether isdepressed as a consequence of aging, further amplifying the deleterioussequelae of additional molecular lesions affecting respiratory chainfunction. Other factors in addition to primary mitochondrial dysfunctionunderlie neurodegeneration in AD, PD, and related disorders. Excitotoxicstimulation and nitric oxide are implicated in both diseases, factorswhich both exacerbate mitochondrial respiratory chain deficits and whosedeleterious actions are exaggerated on a background of respiratory chaindysfunction. Huntington's Disease also involves mitochondrialdysfunction in affected brain regions, with cooperative interactions ofexcitotoxic stimulation and mitochondrial dysfunction contributing toneuronal degeneration. In certain embodiments, sirtuin activatingcompounds may be useful for treating and attenuating progression ofage-related neurodegenerative diseases including AD and PD.

One of the major genetic defects in patients with Amyotrophic LateralSclerosis (ALS or Lou Gehrig's Disease) is mutation or deficiency inCopper-Zinc Superoxide Dismutase (SOD 1), an antioxidant enzyme.Mitochondria both produce and are primary targets for reactive oxygenspecies. Inefficient transfer of electrons to oxygen in mitochondria isthe most significant physiological source of free radicals in mammaliansystems. Deficiencies in antioxidants or antioxidant enzymes can resultin or exacerbate mitochondrial degeneration. Mice transgenic for mutatedSOD1 develop symptoms and pathology similar to those in human ALS. Thedevelopment of the disease in these animals has been shown to involveoxidative destruction of mitochondria followed by functional decline ofmotor neurons and onset of clinical symptoms. Skeletal muscle from ALSpatients has low mitochondrial Complex I activity. In certainembodiments, sirtuin activating compounds may be useful for treatingALS, for reversing or slowing the progression of clinical symptoms.

Oxygen deficiency results in both direct inhibition of mitochondrialrespiratory chain activity by depriving cells of a terminal electronacceptor for Cytochrome c reoxidation at Complex IV, and indirectly,especially in the nervous system, via secondary post-anoxicexcitotoxicity and nitric oxide formation. In conditions like cerebralanoxia, angina or sickle cell anemia crises, tissues are relativelyhypoxic. In such cases, compounds that increase mitochondrial activityprovide protection of affected tissues from deleterious effects ofhypoxia, attenuate secondary delayed cell death, and accelerate recoveryfrom hypoxic tissue stress and injury. In certain embodiments, sirtuinactivating compounds may be useful for preventing delayed cell death(apoptosis in regions like the hippocampus or cortex occurring about 2to 5 days after an episode of cerebral ischemia) after ischemic orhypoxic insult to the brain.

Acidosis due to renal dysfunction is often observed in patients withmitochondrial disease, whether the underlying respiratory chaindysfunction is congenital or induced by ischemia or cytotoxic agentslike cisplatin. Renal tubular acidosis often requires administration ofexogenous sodium bicarbonate to maintain blood and tissue pH. In certainembodiments, sirtuin activating compounds may be useful for treatingrenal tubular acidosis and other forms of renal dysfunction caused bymitochondrial respiratory chain deficits.

During normal aging, there is a progressive decline in mitochondrialrespiratory chain function. Beginning about age 40, there is anexponential rise in accumulation of mitochondrial DNA defects in humans,and a concurrent decline in nuclear-regulated elements of mitochondrialrespiratory activity. Many mitochondrial DNA lesions have a selectionadvantage during mitochondrial turnover, especially in postmitoticcells. The proposed mechanism is that mitochondria with a defectiverespiratory chain produce less oxidative damage to themselves than domitochondria with intact functional respiratory chains (mitochondrialrespiration is the primary source of free radicals in the body).Therefore, normally-functioning mitochondria accumulate oxidative damageto membrane lipids more rapidly than do defective mitochondria, and aretherefore “tagged” for degradation by lysosomes. Since mitochondriawithin cells have a half life of about 10 days, a selection advantagecan result in rapid replacement of functional mitochondria with thosewith diminished respiratory activity, especially in slowly dividingcells. The net result is that once a mutation in a gene for amitochondrial protein that reduces oxidative damage to mitochondriaoccurs, such defective mitochondria will rapidly populate the cell,diminishing or eliminating its respiratory capabilities. Theaccumulation of such cells results in aging or degenerative disease atthe organismal level. This is consistent with the progressive mosaicappearance of cells with defective electron transport activity inmuscle, with cells almost devoid of Cytochrome c Oxidase (COX) activityinterspersed randomly amidst cells with normal activity, and a higherincidence of COX-negative cells in biopsies from older subjects. Theorganism, during aging, or in a variety of mitochondrial diseases, isthus faced with a situation in which irreplaceable postmitotic cells(e.g. neurons, skeletal and cardiac muscle) must be preserved and theirfunction maintained to a significant degree, in the face of aninexorable progressive decline in mitochondrial respiratory chainfunction. Neurons with dysfunctional mitochondria become progressivelymore sensitive to insults like excitotoxic injury. Mitochondrial failurecontributes to most degenerative diseases (especially neurodegeneration)that accompany aging. Congenital mitochondrial diseases often involveearly-onset neurodegeneration similar in fundamental mechanism todisorders that occur during aging of people born with normalmitochondria. In certain embodiments, sirtuin activating compounds maybe useful for treating or attenuating cognitive decline and otherdegenerative consequences of aging.

Mitochondrial DNA damage is more extensive and persists longer thannuclear DNA damage in cells subjected to oxidative stress or cancerchemotherapy agents like cisplatin due to both greater vulnerability andless efficient repair of mitochondrial DNA. Although mitochondrial DNAmay be more sensitive to damage than nuclear DNA, it is relativelyresistant, in some situations, to mutagenesis by chemical carcinogens.This is because mitochondria respond to some types of mitochondrial DNAdamage by destroying their defective genomes rather than attempting torepair them. This results in global mitochondrial dysfunction for aperiod after cytotoxic chemotherapy. Clinical use of chemotherapy agentslike cisplatin, mitomycin, and cytoxan is often accompanied bydebilitating “chemotherapy fatigue”, prolonged periods of weakness andexercise intolerance which may persist even after recovery fromhematologic and gastrointestinal toxicities of such agents. In certainembodiments, sirtuin activating compounds may be useful for treatmentand prevention of side effects of cancer chemotherapy related tomitochondrial dysfunction.

A crucial function of the ovary is to maintain integrity of themitochondrial genome in oocytes, since mitochondria passed onto a fetusare all derived from those present in oocytes at the time of conception.Deletions in mitochondrial DNA become detectable around the age ofmenopause, and are also associated with abnormal menstrual cycles. Sincecells cannot directly detect and respond to defects in mitochondrialDNA, but can only detect secondary effects that affect the cytoplasm,like impaired respiration, redox status, or deficits in pyrimidinesynthesis, such products of mitochondrial function participate as asignal for oocyte selection and follicular atresia, ultimatelytriggering menopause when maintenance of mitochondrial genomic fidelityand functional activity can no longer be guaranteed. This is analogousto apoptosis in cells with DNA damage, which undergo an active processof cellular suicide when genomic fidelity can no longer be achieved byrepair processes. Women with mitochondrial cytopathies affecting thegonads often undergo premature menopause or display primary cyclingabnormalities. Cytotoxic cancer chemotherapy often induces prematuremenopause, with a consequent increased risk of osteoporosis.Chemotherapy-induced amenorrhea is generally due to primary ovarianfailure. The incidence of chemotherapy-induced amenorrhea increases as afunction of age in premenopausal women receiving chemotherapy, pointingtoward mitochondrial involvement. Inhibitors of mitochondrialrespiration or protein synthesis inhibit hormone-induced ovulation, andfurthermore inhibit production of ovarian steroid hormones in responseto pituitary gonadotropins. Women with Down's syndrome typically undergomenopause prematurely, and also are subject to early onset ofAlzheimer-like dementia. Low activity of cytochrome oxidase isconsistently found in tissues of Down's patients and in late-onsetAlzheimer's Disease. Appropriate support of mitochondrial function orcompensation for mitochondrial dysfunction therefore is useful forprotecting against age-related or chemotherapy-induced menopause orirregularities of menstrual cycling or ovulation. In certainembodiments, sirtuin activating compounds may be useful for treating andpreventing amenorrhea, irregular ovulation, menopause, or secondaryconsequences of menopause.

In certain embodiments, sirtuin modulating compounds may be useful fortreatment mitochondrial myopathies. Mitochondrial myopathies range frommild, slowly progressive weakness of the extraocular muscles to severe,fatal infantile myopathies and multisystem encephalomyopathies. Somesyndromes have been defined, with some overlap between them. Establishedsyndromes affecting muscle include progressive external ophthalmoplegia,the Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy,cardiac conduction defects, cerebellar ataxia, and sensorineuraldeafness), the MELAS syndrome (mitochondrial encephalomyopathy, lacticacidosis, and stroke-like episodes), the MERFF syndrome (myoclonicepilepsy and ragged red fibers), limb-girdle distribution weakness, andinfantile myopathy (benign or severe and fatal). Muscle biopsy specimensstained with modified Gomori's trichrome stain show ragged red fibersdue to excessive accumulation of mitochondria. Biochemical defects insubstrate transport and utilization, the Krebs cycle, oxidativephosphorylation, or the respiratory chain are detectable. Numerousmitochondrial DNA point mutations and deletions have been described,transmitted in a maternal, nonmendelian inheritance pattern. Mutationsin nuclear-encoded mitochondrial enzymes occur.

In certain embodiments, sirtuin activating compounds may be useful fortreating patients suffering from toxic damage to mitochondria, such as,toxic damage due to calcium accumulation, excitotoxicity, nitric oxideexposure, drug induced toxic damage, or hypoxia.

A fundamental mechanism of cell injury, especially in excitable tissues,involves excessive calcium entry into cells, as a result of eitherleakage through the plasma membrane or defects in intracellular calciumhandling mechanisms. Mitochondria are major sites of calciumsequestration, and preferentially utilize energy from the respiratorychain for taking up calcium rather than for ATP synthesis, which resultsin a downward spiral of mitochondrial failure, since calcium uptake intomitochondria results in diminished capabilities for energy transduction.

Excessive stimulation of neurons with excitatory amino acids is a commonmechanism of cell death or injury in the central nervous system.Activation of glutamate receptors, especially of the subtype designatedNMDA receptors, results in mitochondrial dysfunction, in part throughelevation of intracellular calcium during excitotoxic stimulation.Conversely, deficits in mitochondrial respiration and oxidativephosphorylation sensitizes cells to excitotoxic stimuli, resulting incell death or injury during exposure to levels of excitotoxicneurotransmitters or toxins that would be innocuous to normal cells.

Nitric oxide (about 1 micromolar) inhibits cytochrome oxidase (ComplexIV) and thereby inhibits mitochondrial respiration; moreover, prolongedexposure to nitric oxide (NO) irreversibly reduces Complex I activity.Physiological or pathophysiological concentrations of NO thereby inhibitpyrimidine biosynthesis. Nitric oxide is implicated in a variety ofneurodegenerative disorders including inflammatory and autoimmunediseases of the central nervous system, and is involved in mediation ofexcitotoxic and post-hypoxic damage to neurons.

Oxygen is the terminal electron acceptor in the respiratory chain.Oxygen deficiency impairs electron transport chain activity, resultingin diminished pyrimidine synthesis as well as diminished ATP synthesisvia oxidative phosphorylation. Human cells proliferate and retainviability under virtually anaerobic conditions if provided with uridineand pynivate (or a similarly effective agent for oxidizing NADH tooptimize glycolytic ATP production).

In certain embodiments, sirtuin activating compounds may be useful fortreating diseases or disorders associated with mitochondrialderegulation.

Transcription of mitochondrial DNA encoding respiratory chain componentsrequires nuclear factors. In neuronal axons, mitochondria must shuttleback and forth to the nucleus in order to maintain respiratory chainactivity. If axonal transport is impaired by hypoxia or by drugs liketaxol which affect microtubule stability, mitochondria distant from thenucleus undergo loss of cytochrome oxidase activity. Accordingly,treatment with a sirtuin activating compound may be useful for promotingnuclear-mitochondrial interactions.

Mitochondria are the primary source of free radicals and reactive oxygenspecies, due to spillover from the mitochondrial respiratory chain,especially when defects in one or more respiratory chain componentsimpairs orderly transfer of electrons from metabolic intermediates tomolecular oxygen. To reduce oxidative damage, cells can compensate byexpressing mitochondrial uncoupling proteins (UCP), of which severalhave been identified. UCP-2 is transcribed in response tooxidative-damage, inflammatory cytokines, or excess lipid loads, e.g.fatty liver and steatohepatitis. UCPs reduce spillover of reactiveoxygen species from mitochondria by discharging proton gradients acrossthe mitochondrial inner membrane, in effect wasting energy produced bymetabolism and rendering cells vulnerable to energy stress as atrade-off for reduced oxidative injury.

Muscle Performance

In other embodiments, the invention provides methods for enhancingmuscle performance by administering a therapeutically effective amountof a sirtuin activating compound. For example, sirtuin activatingcompounds may be useful for improving physical endurance (e.g., abilityto perform a physical task such as exercise, physical labor, sportsactivities, etc.), inhibiting or retarding physical fatigues, enhancingblood oxygen levels, enhancing energy in healthy individuals, enhanceworking capacity and endurance, reducing muscle fatigue, reducingstress, enhancing cardiac and cardiovascular function, improving sexualability, increasing muscle ATP levels, and/or reducing lactic acid inblood. In certain embodiments, the methods involve administering anamount of a sirtuin activating compound that increase mitochondrialactivity, increase mitochondrial biogenesis, and/or increasemitochondrial mass.

Sports performance refers to the ability of the athlete's muscles toperform when participating in sports activities. Enhanced sportsperformance, strength, speed and endurance are measured by an increasein muscular contraction strength, increase in amplitude of musclecontraction, shortening of muscle reaction time between stimulation andcontraction. Athlete refers to an individual who participates in sportsat any level and who seeks to achieve an improved level of strength,speed and endurance in their performance, such as, for example, bodybuilders, bicyclists, long distance runners, short distance runners,etc. An athlete may be hard training, that is, performs sportsactivities intensely more than three days a week or for competition. Anathlete may also be a fitness enthusiast who seeks to improve generalhealth and well-being, improve energy levels, who works out for about1-2 hours about 3 times a week. Enhanced sports performance inmanifested by the ability to overcome muscle fatigue, ability tomaintain activity for longer periods of time, and have a more effectiveworkout.

In the arena of athlete muscle performance, it is desirable to createconditions that permit competition or training at higher levels ofresistance for a prolonged period of time. However, acute and intenseanaerobic use of skeletal muscles often results in impaired athleticperformance, with losses in force and work output, and increased onsetof muscle fatigue, soreness, and dysfunction. It is now recognized thateven a single exhaustive exercise session, or for that matter any acutetrauma to the body such as muscle injury, resistance or exhaustivemuscle exercise, or elective surgery, is characterized by perturbedmetabolism that affects muscle performance in both short and long termphases. Both muscle metabolic/enzymatic activity and gene expression areaffected. For example, disruption of skeletal muscle nitrogen metabolismas well as depletion of sources of metabolic energy occur duringextensive muscle activity. Amino acids, including branched-chain aminoacids, are released from muscles followed by their deamination toelevate serum ammonia and local oxidation as muscle fuel sources, whichaugments metabolic acidosis. In addition, there is a decline incatalytic efficiency of muscle contraction events, as well as analteration of enzymatic activities of nitrogen and energy metabolism.Further, protein catabolism is initiated where rate of protein synthesisis decreased coupled with an increase in the degradation ofnon-contractible protein. These metabolic processes are also accompaniedby free radical generation which further damages muscle cells.

Recovery from fatigue during acute and extended exercise requiresreversal of metabolic and non-metabolic fatiguing factors. Known factorsthat participate in human muscle fatigue, such as lactate, ammonia,hydrogen ion, etc., provide an incomplete and unsatisfactory explanationof the fatigue/recovery process, and it is likely that additionalunknown agents participate (Baker et al., J. Appl. Physiol.74:2294-2300, 1993; Bazzarre et al., J. Am. Coll. Nutr. 11:505-511,1992; Dohm et al., Fed. Proc. 44:348-352, 1985; Edwards In: Biochemistryof Exercise, Proceedings of the Fifth International Symposium on theBiochemistry of Exercise (Kutrgen, Vogel, Poormans, eds.), 1983;MacDougall et al., Acta Physiol. Scand. 146:403-404, 1992; Walser etal., Kidney Int. 32:123-128, 1987). Several studies have also analyzedthe effects of nutritional supplements and herbal supplements inenhancing muscle performance.

Aside from muscle performance during endurance exercise, free radicalsand oxidative stress parameters are affected in pathophysiologicalstates. A substantial body of data now suggests that oxidative stresscontributes to muscle wasting or atrophy in pathophysiological states(reviewed in Clarkson, P. M. Antioxidants and physical performance.Crit. Rev. Food Sci. Nutr. 35: 31-41; 1995; Powers, S. K.; Lennon, S. L.Analysis of cellular responses to free radicals: Focus on exercise andskeletal muscle. Proc. Nutr. Soc. 58: 1025-1033; 1999). For example,with respect to muscular disorders where both muscle endurance andfunction are compensated, the role of nitric oxide (NO), has beenimplicated. In muscular dystrophies, especially those due to defects inproteins that make up the dystrophin-glycoprotein complex (DGC), theenzyme that synthesizes NO, nitric oxide synthase (NOS), has beenassociated. Recent studies of dystrophies related to DGC defects suggestthat one mechanism of cellular injury is functional ischemia related toalterations in cellular NOS and disruption of a normal protective actionof NO. This protective action is the prevention of local ischemia duringcontraction-induced increases in sympathetic vasoconstriction. Rando(Microsc Res Tech 55(4):223-35, 2001), has shown that oxidative injuryprecedes pathologic changes and that muscle cells with defects in theDGC have an increased susceptibility to oxidant challenges. Excessivelipid peroxidation due to free radicals has also been shown to be afactor in myopathic diseases such as McArdle's disease (Russo et al.,Med. Hypotheses. 39(2):147-51, 1992). Furthermore, mitochondrialdysfunction is a well-known correlate of age-related muscle wasting(sarcopenia) and free radical damage has been suggested, though poorlyinvestigated, as a contributing factor (reviewed in Navarro, A.;Lopez-Cepero, J. M.; Sanchez del Pino, M. L. Front. Biosci. 6: D2644;2001). Other indications include acute sarcopenia, for example muscleatrophy and/or cachexia associated with burns, bed rest, limbimmobilization, or major thoracic, abdominal, and/or orthopedic surgery.It is contemplated that the methods of the present invention will alsobe effective in the treatment of muscle related pathological conditions.

In certain embodiments, the invention provides novel dietarycompositions comprising sirtuin modulators, a method for theirpreparation, and a method of using the compositions for improvement ofsports performance. Accordingly, provided are therapeutic compositions,foods and beverages that have actions of improving physical enduranceand/or inhibiting physical fatigues for those people involved inbroadly-defined exercises including sports requiring endurance andlabors requiring repeated muscle exertions. Such dietary compositionsmay additional comprise electrolytes, caffeine, vitamins, carbohydrates,etc.

Other Uses

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used for treating or preventing viralinfections (such as infections by influenza, herpes or papilloma virus)or as antifungal agents. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered as part of a combination drug therapy with anothertherapeutic agent for the treatment of viral diseases, including, forexample, acyclovir, ganciclovir and zidovudine. In another embodiment,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered as part of a combination drugtherapy with another anti-fungal agent including, for example, topicalanti-fungals such as ciclopirox, clotrimazole, econazole, miconazole,nystatin, oxiconazole, terconazole, and tolnaftate, or systemicanti-fungal such as fluconazole (Diflucan), itraconazole (Sporanox),ketoconazole (Nizoral), and miconazole (Monistat I.V.).

Subjects that may be treated as described herein include eukaryotes,such as mammals, e.g., humans, ovines, bovines, equines, porcines,canines, felines, non-human primate, mice, and rats. Cells that may betreated include eukaryotic cells, e.g., from a subject described above,or plant cells, yeast cells and prokaryotic cells, e.g., bacterialcells. For example, modulating compounds may be administered to farmanimals to improve their ability to withstand farming conditions longer.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance, and resistance to apoptosis in plants. In one embodiment, acompound is applied to plants, e.g., on a periodic basis, or to fungi.In another embodiment, plants are genetically modified to produce acompound. In another embodiment, plants and fruits are treated with acompound prior to picking and shipping to increase resistance to damageduring shipping. Plant seeds may also be contacted with compoundsdescribed herein, e.g., to preserve them.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for modulatinglifespan in yeast cells. Situations in which it may be desirable toextend the lifespan of yeast cells include any process in which yeast isused, e.g., the making of beer, yogurt, and bakery items, e.g., bread.Use of yeast having an extended lifespan can result in using less yeastor in having the yeast be active for longer periods of time. Yeast orother mammalian cells used for recombinantly producing proteins may alsobe treated as described herein.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance and resistance to apoptosis in insects. In this embodiment,compounds would be applied to useful insects, e.g., bees and otherinsects that are involved in pollination of plants. In a specificembodiment, a compound would be applied to bees involved in theproduction of honey. Generally, the methods described herein may beapplied to any organism, e.g., eukaryote, that may have commercialimportance. For example, they can be applied to fish (aquaculture) andbirds (e.g., chicken and fowl).

Higher doses of sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may also be used as a pesticide byinterfering with the regulation of silenced genes and the regulation ofapoptosis during development. In this embodiment, a compound may beapplied to plants using a method known in the art that ensures thecompound is bio-available to insect larvae, and not to plants.

At least in view of the link between reproduction and longevity (Longoand Finch, Science, 2002), sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein can be applied to affectthe reproduction of organisms such as insects, animals andmicroorganisms.

4. Assays

Yet other methods contemplated herein include screening methods foridentifying compounds or agents that modulate sirtuins. An agent may bea nucleic acid, such as an aptamer. Assays may be conducted in a cellbased or cell free format. For example, an assay may comprise incubating(or contacting) a sirtuin with a test agent under conditions in which asirtuin can be modulated by an agent known to modulate the sirtuin, andmonitoring or determining the level of modulation of the sirtuin in thepresence of the test agent relative to the absence of the test agent.The level of modulation of a sirtuin can be determined by determiningits ability to deacetylate a substrate. Exemplary substrates areacetylated peptides which can be obtained from BIOMOL (Plymouth Meeting,Pa.). Preferred substrates include peptides of p53, such as thosecomprising an acetylated K382. A particularly preferred substrate is theFluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated peptideArg-His-Lys-Lys. Other substrates are peptides from human histones H3and H4 or an acetylated amino acid (see FIG. 5). Substrates may befluorogenic. The sirtuin may be SIRT1, Sir2, SIRT3, or a portionthereof. For example, recombinant SIRT1 can be obtained from BIOMOL. Thereaction may be conducted for about 30 minutes and stopped, e.g., withnicotinamide. The HDAC fluorescent activity assay/drug discovery kit(AK-500, BIOMOL Research Laboratories) may be used to determine thelevel of acetylation. Similar assays are described in Bitterman et al.(2002) J. Biol. Chem. 277:45099. The level of modulation of the sirtuinin an assay may be compared to the level of modulation of the sirtuin inthe presence of one or more (separately or simultaneously) compoundsdescribed herein, which may serve as positive or negative controls.Sirtuins for use in the assays may be full length sirtuin proteins orportions thereof. Since it has been shown herein that activatingcompounds appear to interact with the N-terminus of SIRT1, proteins foruse in the assays include N-terminal portions of sirtuins, e.g., aboutamino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or 1-252 ofSir2.

In one embodiment, a screening assay comprises (i) contacting a sirtuinwith a test agent and an acetylated substrate under conditionsappropriate for the sirtuin to deacetylate the substrate in the absenceof the test agent; and (ii) determining the level of acetylation of thesubstrate, wherein a lower level of acetylation of the substrate in thepresence of the test agent relative to the absence of the test agentindicates that the test agent stimulates deacetylation by the sirtuin,whereas a higher level of acetylation of the substrate in the presenceof the test agent relative to the absence of the test agent indicatesthat the test agent inhibits deacetylation by the sirtuin.

Methods for identifying an agent that modulates, e.g., stimulates orinhibits, sirtuins in vivo may comprise (i) contacting a cell with atest agent and a substrate that is capable of entering a cell in thepresence of an inhibitor of class I and class II HDACs under conditionsappropriate for the sirtuin to deacetylate the substrate in the absenceof the test agent; and (ii) determining the level of acetylation of thesubstrate, wherein a lower level of acetylation of the substrate in thepresence of the test agent relative to the absence of the test agentindicates that the test agent stimulates deacetylation by the sirtuin,whereas a higher level of acetylation of the substrate in the presenceof the test agent relative to the absence of the test agent indicatesthat the test agent inhibits deacetylation by the sirtuin. A preferredsubstrate is an acetylated peptide, which is also preferablyfluorogenic, as further described herein. The method may furthercomprise lysing the cells to determine the level of acetylation of thesubstrate. Substrates may be added to cells at a concentration rangingfrom about 1 μM to about 10 mM, preferably from about 10 μM to 1 mM,even more preferably from about 100 μM to 1 mM, such as about 200 μM. Apreferred substrate is an acetylated lysine, e.g., ε-acetyl lysine(Fluor de Lys, FdL) or Fluor de Lys-SIRT1. A preferred inhibitor ofclass I and class II HDACs is trichostatin A (TSA), which may be used atconcentrations ranging from about 0.01 to 100 μM, preferably from about0.1 to 10 μM, such as 1 μM. Incubation of cells with the test compoundand the substrate may be conducted for about 10 minutes to 5 hours,preferably for about 1-3 hours. Since TSA inhibits all class I and classII HDACs, and that certain substrates, e.g., Fluor de Lys, is a poorsubstrate for SIRT2 and even less a substrate for SIRT3-7, such an assaymay be used to identify modulators of SIRT1 in vivo.

5. Pharmaceutical Compositions

The sirtuin-modulating compounds described herein may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. For example, sirtuin-modulating compounds andtheir physiologically acceptable salts and solvates may be formulatedfor administration by, for example, injection (e.g. SubQ, IM, IP),inhalation or insufflation (either through the mouth or the nose) ororal, buccal, sublingual, transdermal, nasal, parenteral or rectaladministration. In one embodiment, a sirtuin-modulating compound may beadministered locally, at the site where the target cells are present,i.e., in a specific tissue, organ, or fluid (e.g., blood, cerebrospinalfluid, etc.).

Sirtuin-modulating compounds can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.For parenteral administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the compounds may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets, lozenges, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For administration by inhalation (e.g., pulmonary delivery),sirtuin-modulating compounds may be conveniently delivered in the formof an aerosol spray presentation from pressurized packs or a nebuliser,with the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Sirtuin-modulating compounds may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Sirtuin-modulating compounds may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, sirtuin-modulatingcompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, sirtuin-modulating compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Controlledrelease formula also includes patches.

In certain embodiments, the compounds described herein can be formulatedfor delivery to the central nervous system (CNS) (reviewed in Begley,Pharmacology & Therapeutics 104: 29-45 (2004)). Conventional approachesfor drug delivery to the CNS include: neurosurgical strategies (e.g.,intracerebral injection or intracerebroventricular infusion); molecularmanipulation of the agent (e.g., production of a chimeric fusion proteinthat comprises a transport peptide that has an affinity for anendothelial cell surface molecule in combination with an agent that isitself incapable of crossing the BBB) in an attempt to exploit one ofthe endogenous transport pathways of the BBB; pharmacological strategiesdesigned to increase the lipid solubility of an agent (e.g., conjugationof water-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide).

One possibility to achieve sustained release kinetics is embedding orencapsulating the active compound into nanoparticles. Nanoparticles canbe administrated as powder, as a powder mixture with added excipients oras suspensions. Colloidal suspensions of nanoparticles can easily beadministrated through a cannula with small diameter.

Nanoparticles are particles with a diameter from about 5 nm to up toabout 1000 nm. The term “nanoparticles” as it is used hereinafter refersto particles formed by a polymeric matrix in which the active compoundis dispersed, also known as “nanospheres”, and also refers tonanoparticles which are composed of a core containing the activecompound which is surrounded by a polymeric membrane, also known as“nanocapsules”. In certain embodiments, nanoparticles are preferredhaving a diameter from about 50 nm to about 500 nm, in particular fromabout 100 nm to about 200 nm.

Nanoparticles can be prepared by in situ polymerization of dispersedmonomers or by using preformed polymers. Since polymers prepared in situare often not biodegradable and/or contain toxicological seriousbyproducts, nanoparticles from preformed polymers are preferred.Nanoparticles from preformed polymers can be prepared by differenttechniques, e.g., by emulsion evaporation, solvent displacement,salting-out, mechanical grinding, microprecipitation, and byemulsification diffusion.

With the methods described above, nanoparticles can be formed withvarious types of polymers. For use in the method of the presentinvention, nanoparticles made from biocompatible polymers are preferred.The term “biocompatible” refers to material that after introduction intoa biological environment has no serious effects to the biologicalenvironment. From biocompatible polymers those polymers are especiallypreferred which are also biodegradable. The term “biodegradable” refersto material that after introduction into a biological environment isenzymatically or chemically degraded into smaller molecules, which canbe eliminated subsequently. Examples are polyesters fromhydroxycarboxylic acids such as poly(lactic acid) (PLA), poly(glycolicacid) (PGA), polycaprolactone (PCL), copolymers of lactic acid andglycolic acid (PLGA), copolymers of lactic acid and caprolactone,polyepsilon caprolactone, polyhyroxy butyric acid and poly(ortho)esters,polyurethanes, polyanhydrides, polyacetals, polydihydropyrans,polycyanoacrylates, natural polymers such as alginate and otherpolysaccharides including dextran and cellulose, collagen and albumin.

Suitable surface modifiers can preferably be selected from known organicand inorganic pharmaceutical excipients. Such excipients include variouspolymers, low molecular weight oligomers, natural products andsurfactants. Preferred surface modifiers include nonionic and ionicsurfactants. Representative examples of surface modifiers includegelatin, casein, lecithin (phosphatides), gum acacia, cholesterol,tragacanth, stearic acid, benzalkonium chloride, calcium stearate,glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifyingwax, sorbitan esters, polyoxyethylene alkyl ethers, e.g., macrogolethers such as cetomacrogol 1000, polyoxyethylene castor oilderivatives, polyoxyethylene sorbitan fatty acid esters, e.g., thecommercially available Tweens™, polyethylene glycols, polyoxyethylenestearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate,carboxymethylcellulose calcium, carboxymethylcellulose sodium,methylcellulose, hydroxyethylcellulose, hydroxy propylcellulose,hydroxypropylmethylcellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, andpolyvinylpyrrolidone (PVP). Most of these surface modifiers are knownpharmaceutical excipients and are described in detail in the Handbook ofPharmaceutical Excipients, published jointly by the AmericanPharmaceutical Association and The Pharmaceutical Society of GreatBritain, the Pharmaceutical Press, 1986.

Further description on preparing nanoparticles can be found, forexample, in U.S. Pat. No. 6,264,922, the contents of which areincorporated herein by reference.

Liposomes are a further drug delivery system which is easily injectable.Accordingly, in the method of invention the active compounds can also beadministered in the form of a liposome delivery system. Liposomes arewell-known by a person skilled in the art. Liposomes can be formed froma variety of phospholipids, such as cholesterol, stearylamine ofphosphatidylcholines. Liposomes being usable for the method of inventionencompass all types of liposomes including, but not limited to, smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles.

Liposomes are used for a variety of therapeutic purposes, and inparticular, for carrying therapeutic agents to target cells.Advantageously, liposome-drug formulations offer the potential ofimproved drug-delivery properties, which include, for example,controlled drug release. An extended circulation time is often neededfor liposomes to reach a target region, cell or site. In particular,this is necessary where the target region, cell or site is not locatednear the site of administration. For example, when liposomes areadministered systemically, it is desirable to coat the liposomes with ahydrophilic agent, for example, a coating of hydrophilic polymer chainssuch as polyethylene glycol (PEG) to extend the blood circulationlifetime of the liposomes. Such surface-modified liposomes are commonlyreferred to as “long circulating” or “sterically stabilized” liposomes.

One surface modification to a liposome is the attachment of PEG chains,typically having a molecular weight from about 1000 daltons (Da) toabout 5000 Da, and to about 5 mole percent (%) of the lipids making upthe liposomes (see, for example, Stealth Liposomes, CRC Press, Lasic, D.and Martin, F., eds., Boca Raton, Fla., (1995)), and the citedreferences therein. The pharmacokinetics exhibited by such liposomes arecharacterized by a dose-independent reduction in uptake of liposomes bythe liver and spleen via the mononuclear phagocyte system (MPS), andsignificantly prolonged blood circulation time, as compared tonon-surface-modified liposomes, which tend to be rapidly removed fromthe blood and accumulated in the liver and spleen.

In certain embodiments, the complex is shielded to increase thecirculatory half-life of the complex or shielded to increase theresistance of nucleic acid to degradation, for example degradation bynucleases.

As used herein, the term “shielding”, and its cognates such as“shielded”, refers to the ability of “shielding moieties” to reduce thenon-specific interaction of the complexes described herein with serumcomplement or with other species present in serum in vitro or in vivo.Shielding moieties may decrease the complex interaction with or bindingto these species through one or more mechanisms, including, for example,non-specific steric or non-specific electronic interactions. Examples ofsuch interactions include non-specific electrostatic interactions,charge interactions, Van der Waals interactions, steric-hindrance andthe like. For a moiety to act as a shielding moiety, the mechanism ormechanisms by which it may reduce interaction with, association with orbinding to the serum complement or other species does not have to beidentified. One can determine whether a moiety can act as a shieldingmoiety by determining whether or to what extent a complex binds serumspecies.

It should be noted that “shielding moieties” can be multifunctional. Forexample, a shielding moiety may also function as, for example, atargeting factor. A shielding moiety may also be referred to asmultifunctional with respect to the mechanism(s) by which it shields thecomplex. While not wishing to be limited by proposed mechanism ortheory, examples of such a multifunctional shielding moiety are pHsensitive endosomal membrane-disruptive synthetic polymers, such as PPAAor PEAA. Certain poly(alkylacrylic acids) have been shown to disruptendosomal membranes while leaving the outer cell surface membrane intact(Stayton et al. (2000) J. Controll. Release 65:203-220; Murthy et al.(1999) J. Controll. Release 61:137-143; WO 99/34831), thereby increasingcellular bioavailability and functioning as a targeting factor. However,PPAA reduces binding of serum complement to complexes in which it isincorporated, thus functioning as a shielding moiety.

Another way to produce a formulation, particularly a solution, of asirtuin modulator such as resveratrol or a derivative thereof, isthrough the use of cyclodextrin. By cyclodextrin is meant α-, β-, orγ-cyclodextrin. Cyclodextrins are described in detail in Pitha et al.,U.S. Pat. No. 4,727,064, which is incorporated herein by reference.Cyclodextrins are cyclic oligomers of glucose; these compounds forminclusion complexes with any drug whose molecule can fit into thelipophile-seeking cavities of the cyclodextrin molecule.

The cyclodextrin of the compositions according to the invention may beα-, β-, or γ-cyclodextrin. α-cyclodextrin contains six glucopyranoseunits; β-cyclodextrin contains seven glucopyranose units; andγ-cyclodextrin contains eight glucopyranose units. The molecule isbelieved to form a truncated cone having a core opening of 4.7-5.3angstroms, 6.0-6.5 angstroms, and 7.5-8.3 angstroms in α-, β-, orγ-cyclodextrin respectively. The composition according to the inventionmay comprise a mixture of two or more of the α-, β-, or γ-cyclodextrins.Typically, however, the composition according to the invention willcomprise only one of the α-, β-, or γ-cyclodextrins.

Most preferred cyclodextrins in the compositions according to theinvention are amorphous cyclodextrin compounds. By amorphouscyclodextrin is meant non-crystalline mixtures of cyclodextrins whereinthe mixture is prepared from α-, β-, or γ-cyclodextrin. In general, theamorphous cyclodextrin is prepared by non-selective alkylation of thedesired cyclodextrin species. Suitable alkylation agents for thispurpose include but are not limited to propylene oxide, glycidol,iodoacetamide, chloroacetate, and 2-diethylaminoethlychloride. Reactionsare carried out to yield mixtures containing a plurality of componentsthereby preventing crystallization of the cyclodextrin. Variousalkylated cyclodextrins can be made and of course will vary, dependingupon the starting species of cyclodextrin and the alkylating agent used.Among the amorphous cyclodextrins suitable for compositions according tothe invention are hydroxypropyl, hydroxyethyl, glucosyl, maltosyl andmaltotriosyl derivatives of β-cyclodextrin,carboxyamidomethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin,hydroxypropyl-β-cyclodextrin and diethylamino-β-cyclodextrin.

One example of resveratrol dissolved in the presence of a cyclodextrinis provided in Marier et al., J. Pharmacol. Exp. Therap. 302:369-373(2002), the contents of which are incorporated herein by reference,where a 6 mg/mL solution of resveratrol was prepared using 0.9% salinecontaining 20% hydroxylpropyl-β-cyclodextrin.

As mentioned above, the compositions of matter of the invention comprisean aqueous preparation of preferably substituted amorphous cyclodextrinand one or more sirtuin modulators. The relative amounts of sirtuinmodulators and cyclodextrin will vary depending upon the relative amountof each of the sirtuin modulators and the effect of the cyclodextrin onthe compound. In general, the ratio of the weight of compound of thesirtuin modulators to the weight of cyclodextrin compound will be in arange between 1:1 and 1:100. A weight to weight ratio in a range of 1:5to 1:50 and more preferably in a range of 1:10 to 1:20 of the compoundselected from sirtuin modulators to cyclodextrin are believed to be themost effective for increased circulating availability of the sirtuinmodulator.

Importantly, if the aqueous solution comprising the sirtuin modulatorsand a cyclodextrin is to be administered parenterally, especially viathe intravenous route, a cyclodextrin will be substantially free ofpyrogenic contaminants. Various forms of cyclodextrin, such as forms ofamorphous cyclodextrin, may be purchased from a number of vendorsincluding Sigma-Aldrich, Inc. (St. Louis, Mo., USA). A method for theproduction of hydroxypropyl-β-cyclodextrin is disclosed in Pitha et al.,U.S. Pat. No. 4,727,064 which is incorporated herein by reference.

Additional description of the use of cyclodextrin for solubilizingcompounds can be found in US 2005/0026849, the contents of which areincorporated herein by reference.

Rapidly disintegrating or dissolving dosage forms are useful for therapid absorption, particularly buccal and sublingual absorption, ofpharmaceutically active agents. Fast melt dosage forms are beneficial topatients, such as aged and pediatric patients, who have difficulty inswallowing typical solid dosage forms, such as caplets and tablets.Additionally, fast melt dosage forms circumvent drawbacks associatedwith, for example, chewable dosage forms, wherein the length of time anactive agent remains in a patient's mouth plays an important role indetermining the amount of taste masking and the extent to which apatient may object to throat grittiness of the active agent.

To overcome such problems manufacturers have developed a number of fastmelt solid dose oral formulations. These are available frommanufacturers including Cima Labs, Fuisz Technologies Ltd., Prographarm,R. P. Scherer, Yamanouchi-Shaklee, and McNeil-PPC, Inc. All of thesemanufacturers market different types of rapidly dissolving solid oraldosage forms. See e.g., patents and publications by Cima Labs such asU.S. Pat. Nos. 5,607,697, 5,503,846, 5,223,264, 5,401,513, 5,219,574,and 5,178,878, WO 98/46215, WO 98/14179; patents to Fuisz Technologies,now part of BioVail, such as U.S. Pat. Nos. 5,871,781, 5,869,098,5,866,163, 5,851,553, 5,622,719, 5,567,439, and 5,587,172; U.S. Pat. No.5,464,632 to Prographarm; patents to R. P. Scherer such as U.S. Pat.Nos. 4,642,903, 5,188,825, 5,631,023 and 5,827,541; patents toYamanouchi-Shaklee such as U.S. Pat. Nos. 5,576,014 and 5,446,464;patents to Janssen such as U.S. Pat. Nos. 5,807,576, 5,635,210,5,595,761, 5,587,180 and 5,776,491; U.S. Pat. Nos. 5,639,475 and5,709,886 to Eurand America, Inc.; U.S. Pat. Nos. 5,807,578 and5,807,577 to L.A.B. Pharmaceutical Research; patents to ScheringCorporation such as U.S. Pat. Nos. 5,112,616 and 5,073,374; U.S. Pat.No. 4,616,047 to Laboratoire L. LaFon; U.S. Pat. No. 5,501,861 to TakedaChemicals Inc., Ltd.; and U.S. Pat. No. 6,316,029 to Elan.

In one example of fast melt tablet preparation, granules for fast melttablets made by either the spray drying or pre-compacting processes aremixed with excipients and compressed into tablets using conventionaltablet making machinery. The granules can be combined with a variety ofcarriers including low density, high moldability saccharides, lowmoldability saccharides, polyol combinations, and then directlycompressed into a tablet that exhibits an improved dissolution anddisintegration profile.

The tablets according to the present invention typically have a hardnessof about 2 to about 6 Strong-Cobb units (scu). Tablets within thishardness range disintegrate or dissolve rapidly when chewed.Additionally, the tablets rapidly disentegrate in water. On average, atypical 1.1 to 1.5 gram tablet disintegrates in 1-3 minutes withoutstirring. This rapid disintegration facilitates delivery of the activematerial.

The granules used to make the tablets can be, for example, mixtures oflow density alkali earth metal salts or carbohydrates. For example, amixture of alkali earth metal salts includes a combination of calciumcarbonate and magnesium hydroxide. Similarly, a fast melt tablet can beprepared according to the methods of the present invention thatincorporates the use of A) spray dried extra light calciumcarbonate/maltodextrin, B) magnesium hydroxide and C) a eutectic polyolcombination including Sorbitol Instant, xylitol and mannitol. Thesematerials have been combined to produce a low density tablet thatdissolves very readily and promotes the fast disintegration of theactive ingredient. Additionally, the pre-compacted and spray driedgranules can be combined in the same tablet.

For fast melt tablet preparation, a sirtuin modulator useful in thepresent invention can be in a form such as solid, particulate, granular,crystalline, oily or solution. The sirtuin modulator for use in thepresent invention may be a spray dried product or an adsorbate that hasbeen pre-compacted to a harder granular form that reduces the medicamenttaste. A pharmaceutical active ingredient for use in the presentinvention may be spray dried with a carrier that prevents the activeingredient from being easily extracted from the tablet when chewed.

In addition to being directly added to the tablets of the presentinvention, the medicament drug itself can be processed by thepre-compaction process to achieve an increased density prior to beingincorporated into the formulation.

The pre-compaction process used in the present invention can be used todeliver poorly soluble pharmaceutical materials so as to improve therelease of such pharmaceutical materials over traditional dosage forms.This could allow for the use of lower dosage levels to deliverequivalent bioavailable levels of drug and thereby lower toxicity levelsof both currently marketed drug and new chemical entities. Poorlysoluble pharmaceutical materials can be used in the form ofnanoparticles, which are nanometer-sized particles.

In addition to the active ingredient and the granules prepared from lowdensity alkali earth metal salts and/or water soluble carbohydrates, thefast melt tablets can be formulated using conventional carriers orexcipients and well established pharmaceutical techniques. Conventionalcarriers or excipients include, but are not limited to, diluents,binders, adhesives (i.e., cellulose derivatives and acrylicderivatives), lubricants (i.e., magnesium or calcium stearate, vegetableoils, polyethylene glycols, talc, sodium lauryl sulphate, polyoxyethylene monostearate), disintegrants, colorants, flavorings,preservatives, sweeteners and miscellaneous materials such as buffersand adsorbents.

Additional description of the preparation of fast melt tablets can befound, for example, in U.S. Pat. No. 5,939,091, the contents of whichare incorporated herein by reference.

Pharmaceutical compositions (including cosmetic preparations) maycomprise from about 0.00001 to 100% such as from 0.001 to 10% or from0.1% to 5% by weight of one or more sirtuin-modulating compoundsdescribed herein.

In one embodiment, a sirtuin-modulating compound described herein, isincorporated into a topical formulation containing a topical carrierthat is generally suited to topical drug administration and comprisingany such material known in the art. The topical carrier may be selectedso as to provide the composition in the desired form, e.g., as anointment, lotion, cream, microemulsion, gel, oil, solution, or the like,and may be comprised of a material of either naturally occurring orsynthetic origin. It is preferable that the selected carrier notadversely affect the active agent or other components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like.

Formulations may be colorless, odorless ointments, lotions, creams,microemulsions and gels.

Sirtuin-modulating compounds may be incorporated into ointments, whichgenerally are semisolid preparations which are typically based onpetrolatum or other petroleum derivatives. The specific ointment base tobe used, as will be appreciated by those skilled in the art, is one thatwill provide for optimum drug delivery, and, preferably, will providefor other desired characteristics as well, e.g., emolliency or the like.As with other carriers or vehicles, an ointment base should be inert,stable, nonirritating and nonsensitizing. As explained in Remington's(supra) ointment bases may be grouped in four classes: oleaginous bases;emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginousointment bases include, for example, vegetable oils, fats obtained fromanimals, and semisolid hydrocarbons obtained from petroleum.Emulsifiable ointment bases, also known as absorbent ointment bases,contain little or no water and include, for example, hydroxystearinsulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointmentbases are either water-in-oil (W/O) emulsions or oil-in-water (O/W)emulsions, and include, for example, cetyl alcohol, glycerylmonostearate, lanolin and stearic acid. Exemplary water-soluble ointmentbases are prepared from polyethylene glycols (PEGs) of varying molecularweight; again, reference may be had to Remington's, supra, for furtherinformation.

Sirtuin-modulating compounds may be incorporated into lotions, whichgenerally are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semiliquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and maycomprise a liquid oily emulsion of the oil-in-water type. Lotions arepreferred formulations for treating large body areas, because of theease of applying a more fluid composition. It is generally necessarythat the insoluble matter in a lotion be finely divided. Lotions willtypically contain suspending agents to produce better dispersions aswell as compounds useful for localizing and holding the active agent incontact with the skin, e.g., methylcellulose, sodiumcarboxymethylcellulose, or the like. An exemplary lotion formulation foruse in conjunction with the present method contains propylene glycolmixed with a hydrophilic petrolatum such as that which may be obtainedunder the trademark Aquaphor® from Beiersdorf, Inc. (Norwalk, Conn.).

Sirtuin-modulating compounds may be incorporated into creams, whichgenerally are viscous liquid or semisolid emulsions, either oil-in-wateror water-in-oil. Cream bases are water-washable, and contain an oilphase, an emulsifier and an aqueous phase. The oil phase is generallycomprised of petrolatum and a fatty alcohol such as cetyl or stearylalcohol; the aqueous phase usually, although not necessarily, exceedsthe oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation, as explained in Remington's, supra,is generally a nonionic, anionic, cationic or amphoteric surfactant.

Sirtuin-modulating compounds may be incorporated into microemulsions,which generally are thermodynamically stable, isotropically cleardispersions of two immiscible liquids, such as oil and water, stabilizedby an interfacial film of surfactant molecules (Encyclopedia ofPharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9).For the preparation of microemulsions, surfactant (emulsifier),co-surfactant (co-emulsifier), an oil phase and a water phase arenecessary. Suitable surfactants include any surfactants that are usefulin the preparation of emulsions, e.g., emulsifiers that are typicallyused in the preparation of creams. The co-surfactant (or “co-emulsifer”)is generally selected from the group of polyglycerol derivatives,glycerol derivatives and fatty alcohols. Preferredemulsifier/co-emulsifier combinations are generally although notnecessarily selected from the group consisting of: glyceryl monostearateand polyoxyethylene stearate; polyethylene glycol and ethylene glycolpalmitostearate; and caprilic and capric triglycerides and oleoylmacrogolglycerides. The water phase includes not only water but also,typically, buffers, glucose, propylene glycol, polyethylene glycols,preferably lower molecular weight polyethylene glycols (e.g., PEG 300and PEG 400), and/or glycerol, and the like, while the oil phase willgenerally comprise, for example, fatty acid esters, modified vegetableoils, silicone oils, mixtures of mono- di- and triglycerides, mono- anddi-esters of PEG (e.g., oleoyl macrogol glycerides), etc.

Sirtuin-modulating compounds may be incorporated into gel formulations,which generally are semisolid systems consisting of either suspensionsmade up of small inorganic particles (two-phase systems) or largeorganic molecules distributed substantially uniformly throughout acarrier liquid (single phase gels). Single phase gels can be made, forexample, by combining the active agent, a carrier liquid and a suitablegelling agent such as tragacanth (at 2 to 5%), sodium alginate (at2-10%), gelatin (at 2-15%), methylcellulose (at 3-5%), sodiumcarboxymethylcellulose (at 2-5%), carbomer (at 0.3-5%) or polyvinylalcohol (at 10-20%) together and mixing until a characteristic semisolidproduct is produced. Other suitable gelling agents includemethylhydroxycellulose, polyoxyethylene-polyoxypropylene,hydroxyethylcellulose and gelatin. Although gels commonly employ aqueouscarrier liquid, alcohols and oils can be used as the carrier liquid aswell.

Various additives, known to those skilled in the art, may be included informulations, e.g., topical formulations. Examples of additives include,but are not limited to, solubilizers, skin permeation enhancers,opacifiers, preservatives (e.g., anti-oxidants), gelling agents,buffering agents, surfactants (particularly nonionic and amphotericsurfactants), emulsifiers, emollients, thickening agents, stabilizers,humectants, colorants, fragrance, and the like. Inclusion ofsolubilizers and/or skin permeation enhancers is particularly preferred,along with emulsifiers, emollients and preservatives. An optimum topicalformulation comprises approximately: 2 wt. % to 60 wt. %, preferably 2wt. % to 50 wt. %, solubilizer and/or skin permeation enhancer; 2 wt. %to 50 wt. %, preferably 2 wt. % to 20 wt. %, emulsifiers; 2 wt. % to 20wt. % emollient; and 0.01 to 0.2 wt. % preservative, with the activeagent and carrier (e.g., water) making of the remainder of theformulation.

A skin permeation enhancer serves to facilitate passage of therapeuticlevels of active agent to pass through a reasonably sized area ofunbroken skin. Suitable enhancers are well known in the art and include,for example: lower alkanols such as methanol ethanol and 2-propanol;alkyl methyl sulfoxides such as dimethylsulfoxide (DMSO),decylmethylsulfoxide (C₁₀ MSO) and tetradecylmethyl sulfboxide;pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone andN-(-hydroxyethyl)pyrrolidone; urea; N,N-diethyl-m-toluamide; C₂-C₆alkanediols; miscellaneous solvents such as dimethyl formamide (DMF),N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol; and the1-substituted azacycloheptan-2-ones, particularly1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under thetrademark Azone® from Whitby Research Incorporated, Richmond, Va.).

Examples of solubilizers include, but are not limited to, the following:hydrophilic ethers such as diethylene glycol monoethyl ether(ethoxydiglycol, available commercially as Transcutol®) and diethyleneglycol monoethyl ether oleate (available commercially as Softcutol®);polyethylene castor oil derivatives such as polyoxy 35 castor oil,polyoxy 40 hydrogenated castor oil, etc.; polyethylene glycol,particularly lower molecular weight polyethylene glycols such as PEG 300and PEG 400, and polyethylene glycol derivatives such as PEG-8caprylic/capric glycerides (available commercially as Labrasol®); alkylmethyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone andN-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act asabsorption enhancers. A single solubilizer may be incorporated into theformulation, or a mixture of solubilizers may be incorporated therein.

Suitable emulsifiers and co-emulsifiers include, without limitation,those emulsifiers and co-emulsifiers described with respect tomicroemulsion formulations. Emollients include, for example, propyleneglycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2)myristyl ether propionate, and the like.

Other active agents may also be included in formulations, e.g., otheranti-inflammatory agents, analgesics, antimicrobial agents, antifungalagents, antibiotics, vitamins, antioxidants, and sunblock agentscommonly found in sunscreen formulations including, but not limited to,anthranilates, benzophenones (particularly benzophenone-3), camphorderivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid(PABA) and derivatives thereof, and salicylates (e.g., octylsalicylate).

In certain topical formulations, the active agent is present in anamount in the range of approximately 0.25 wt. % to 75 wt. % of theformulation, preferably in the range of approximately 0.25 wt. % to 30wt. % of the formulation, more preferably in the range of approximately0.5 wt. % to 15 wt. % of the formulation, and most preferably in therange of approximately 1.0 wt. % to 10 wt. % of the formulation.

Topical skin treatment compositions can be packaged in a suitablecontainer to suit its viscosity and intended use by the consumer. Forexample, a lotion or cream can be packaged in a bottle or a roll-ballapplicator, or a propellant-driven aerosol device or a container fittedwith a pump suitable for finger operation. When the composition is acream, it can simply be stored in a non-deformable bottle or squeezecontainer, such as a tube or a lidded jar. The composition may also beincluded in capsules such as those described in U.S. Pat. No. 5,063,507.Accordingly, also provided are closed containers containing acosmetically acceptable composition as herein defined.

In an alternative embodiment, a pharmaceutical formulation is providedfor oral or parenteral administration, in which case the formulation maycomprises a modulating compound-containing microemulsion as describedabove, but may contain alternative pharmaceutically acceptable carriers,vehicles, additives, etc. particularly suited to oral or parenteral drugadministration. Alternatively, a modulating compound-containingmicroemulsion may be administered orally or parenterally substantiallyas described above, without modification.

Phospholipids complexes, e.g., resveratrol-phospholipid complexes, andtheir preparation are described in U.S. Patent Application PublicationNo. 2004/116386. Methods for stabilizing active components usingpolyol/polymer microcapsules, and their preparation are described inUS20040108608. Processes for dissolving lipophilic compounds in aqueoussolution with amphiphilic block copolymers are described in WO04/035013.

Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of a sirtuin-modulating compound, or byinsertion of a sustained release device that releases asirtuin-modulating compound. A sirtuin-modulating compound thatincreases or decreases the level and/or activity of a sirtuin proteinmay be delivered in a pharmaceutically acceptable ophthalmic vehicle,such that the compound is maintained in contact with the ocular surfacefor a sufficient time period to allow the compound to penetrate thecorneal and internal regions of the eye, as for example the anteriorchamber, posterior chamber, vitreous body, aqueous humor, vitreoushumor, cornea, iris/ciliary, lens, choroid/retina and sclera. Thepharmaceutically-acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material. Alternatively, thecompounds of the invention may be injected directly into the vitreousand aqueous humour. In a further alternative, the compounds may beadministered systemically, such as by intravenous infusion or injection,for treatment of the eye.

Sirtuin-modulating compounds described herein may be stored in oxygenfree environment according to methods in the art. For example,resveratrol or analog thereof can be prepared in an airtight capsule fororal administration, such as Capsugel from Pfizer, Inc.

Cells, e.g., treated ex vivo with a sirtuin-modulating compound, can beadministered according to methods for administering a graft to asubject, which may be accompanied, e.g., by administration of animmunosuppressant drug, e.g., cyclosporin A. For general principles inmedicinal formulation, the reader is referred to Cell Therapy: Stem CellTransplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn& W. Sheridan eds, Cambridge University Press, 1996; and HematopoieticStem Cell Therapy, E. D. Ball, J. Lister & P. Law, ChurchillLivingstone, 2000.

Toxicity and therapeutic efficacy of sirtuin-modulating compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals. The LD₅₀ is the dose lethal to 50% of thepopulation. The ED₅₀ is the dose therapeutically effective in 50% of thepopulation. The dose ratio between toxic and therapeutic effects(LD₅₀/ED₅₀) is the therapeutic index. Sirtuin-modulating compounds thatexhibit large therapeutic indexes are preferred. Whilesirtuin-modulating compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

6. Kits

Also provided herein are kits, e.g., kits for therapeutic purposes orkits for modulating the lifespan of cells or modulating apoptosis. A kitmay comprise one or more sirtuin-modulating compounds, e.g., inpremeasured doses. A kit may optionally comprise devices for contactingcells with the compounds and instructions for use. Devices includesyringes, stents and other devices for introducing a sirtuin-modulatingcompound into a subject (e.g., the blood vessel of a subject) orapplying it to the skin of a subject.

Another type of kit contemplated by the invention are kits foridentifying sirtuin-modulating compounds. Such kits contain (1) asirtuin or sirtuin-containing material and (2) a sirtuin-modulatingcompound of the invention, which are in separate vessels. Such kits canbe used, for example, to perform a competition-type assay to test othercompounds (typically provided by the user) for sirtuin-modulatingactivity. In certain embodiments, these kits further comprise means fordetermining sirtuin activity (e.g., a peptide with an appropriateindicator, such as those disclosed in the Exemplification).

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Identification of Sirtuin Modulators Using SIRT1

A fluorescence polarization or mass spectrometry based assay was used toidentify modulators of SIRT1 activity. The same assay may be used toidentify modulators of any sirtuin protein. The fluorescencepolarization assays utilizes one of two different peptides based on afragment of p53, a known sirtuin deacetylation target. Compounds weretested using a substrate containing peptide 1 having 20 amino acidresidues as follows:Ac-EE-K(biotin)-GQSTSSHSK(Ac)NleSTEG-K(MR121)-EE-NH₂ (SEQ ID NO: 1)wherein K(biotin) is a biotinolated lysine residue, K(Ac) is anacetylated lysine residue, Nle is norleucine and K(MR121) is a lysineresidue modified by an MR121 fluorophore. This peptide is labeled withthe fluorophore MR121 (excitation 635 nm/emission 680 nm) at theC-termini and biotin at the N-termini. The sequence of the peptidesubstrates are based on p53 with several modifications. In particular,all arginine and leucine residues other than the acetylated lysineresidues have replaced with serine so that the peptides are notsusceptible to trypsin cleavage in the absence of deacetylation. Inaddition, the methionine residues naturally present in the sequenceshave been replaced with the norleucine because the methionine may besusceptible to oxidation during synthesis and purification. As analternative substrate in the assay, the following peptide 2 has alsobeen used: Ac-EE-K(biotin)-GQSTSSHSK(Ac)NleSTEG-K(5TMR)-EE-NH2 (SEQ IDNO: 2) wherein K(Ac) is an acetylated lysine residue and Nle is anorleucine. The peptide is labeled with the fluorophore 5TMR (excitation540 nm/emission 580 nm) at the C-terminus. The sequence of the peptidesubstrate is also based on p53 with several modifications. In addition,the methionine residue naturally present in the sequence was replacedwith the norleucine because the methionine may be susceptible tooxidation during synthesis and purification.

The peptide substrates were exposed to a sirtuin protein in the presenceof NAD⁺ to allow deacetylation of the substrate and render it sensitiveto cleavage by trypsin. Trypsin was then added and the reaction wascarried to completion (i.e., the deacetylated substrate is cleaved)releasing the MR121 or 5TMR fragment. Streptavidin is then added to thereaction where it can bind both the uncleaved substrate (i.e., anyremaining acetylated substrate) and the non-fluorescent portion of thecleaved peptide substrate (i.e., the biotin containing fragment). Thefluorescence polarization signal observed for the full length peptidesubstrates bound to streptavidin was higher than the fluorescencepolarization signal observed for the released MR121 or 5TMR C-terminalfragment. In this way, the fluorescence polarization obtained isinversely proportional to the level of deacetylation (e.g., the signalis inversely proportional to the activity of the sirtuin protein).Results were read on a microplate fluorescence polarization reader(Molecular Devices Spectramax MD) with appropriate excitation andemission filters.

The fluorescence polarization assays using peptide 1 is conducted asfollows: 0.5 μM peptide substrate and 150 μM βNAD⁺ is incubated with 0.1μg/mL of SIRT1 for 60 minutes at 37° C. in a reaction buffer (25 mMTris-acetate pH8, 137 mM Na—Ac, 2.7 mM K—Ac, 1 mM Mg—Ac, 0.05% Tween-20,0.1% Pluronic F127, 10 mM CaCl₂, 5 mM DTT, 0.025% BSA, 0.15 mMNicotinamide). Test compounds were solubilized in DMSO and added to thereaction at 11 concentrations ranging from 0.7 μM to 100 μM.

Fluorescence polarization assays using peptide 2 is conducted asfollows: 0.5 μM peptide substrate and 120 μM βNAD⁺ were incubated with 3nM SIRT1 for 20 minutes at 25° C. in a reaction buffer (25 mMTris-acetate pH8, 137 mM Na—Ac, 2.7 mM K—Ac, 1 mM Mg—Ac, 0.05% Tween-20,0.1% Pluronic F127, 10 mM CaCl₂, 5 mM DTT, 0.025% BSA). Test compounds19-56 were solubilized in DMSO and added to the reaction at 10concentrations ranging from 300 μM to 0.15 μM in three-fold dilutions.

After the incubation with SIRT1, nicotinamide was added to the reactionto a final concentration of 3 mM to stop the deacetylation reaction and0.5 μg/mL of trypsin was added to cleave the deacetylated substrate. Thereaction was incubated for 30 minutes at 37° C. in the presence of 1 μMstreptavidin. Fluorescent polarization was determined at excitation (650nm) and emission (680 nm) wavelengths. The level of activity of thesirtuin protein in the presence of the various concentrations of testcompound is then determined and may be compared to the level of activityof the sirtuin protein in the absence of the test compound, and/or thelevel of activity of the sirtuin proteins in the negative control (e.g.,level of inhibition) and positive control (e.g., level of activation)described below.

For the Fluorescence Polarization assays, a control for inhibition ofsirtuin activity is conducted by adding 1 μL of 500 mM nicotinamide as anegative control at the start of the reaction (e.g., permitsdetermination of maximum sirtuin inhibition). A control for activationof sirtuin activity was conducted using 3 nM of sirtuin protein, with 1μL of DMSO in place of compound, to reach baseline deacetylation of thesubstrate (e.g., to determine normalized sirtuin activity).

The mass spectrometry based assay utilizes a peptide having 20 aminoacid residues as follows:Ac-EE-K(biotin)-GQSTSSHS-K(Ac)-Nle-STEG-K(5TMR)-EE-NH₂ (SEQ ID NO: 2)wherein K(Ac) is an acetylated lysine residue and Nle is a norleucine.The peptide is labeled with the fluorophore 5TMR (excitation 540nm/emission 580 inn) at the C-terminus. The sequence of the peptidesubstrate is based on p53 with several modifications. In addition, themethionine residue naturally present in the sequence was replaced withthe norleucine because the methionine may be susceptible to oxidationduring synthesis and purification.

The mass spectrometry assay is conducted as follows: 0.5 μM peptidesubstrate and 120 μM βNAD⁺ is incubated with 10 nM SIRT1 for 25 minutesat 25° C. in a reaction buffer (50 mM Tris-acetate pH 8, 137 mM NaCl,2.7 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.05% BSA). Test compounds may beadded to the reaction as described above. The SirT1 gene is cloned intoa T7-promoter containing vector and transformed into BL21 (DE3). Afterthe 25 minute incubation with SIRT1, 10 μL of 10% formic acid is addedto stop the reaction. Reactions are sealed and frozen for later massspec analysis. Determination of the mass of the substrate peptide allowsfor precise determination of the degree of acetylation (i.e. startingmaterial) as compared to deacetylated peptide (product).

For the mass spectrometry based assay, a control for inhibition ofsirtuin activity is conducted by adding 1 μL of 500 mM nicotinamide as anegative control at the start of the reaction (e.g., permitsdetermination of maximum sirtuin inhibition). A control for activationof sirtuin activity is conducted using 10 nM of sirtuin protein, with 1μL of DMSO in place of compound, to determine the amount ofdeacetylation of the substrate at a given timepoint within the linearrange of the assay. This timepoint is the same as that used for testcompounds and, within the linear range, the endpoint represents a changein velocity.

For each of the above assays, SIRT1 protein was expressed and purifiedas follows. The SirT1 gene was cloned into a T7-promoter containingvector and transformed into BL21(DE3). The protein was expressed byinduction with 1 mM IPTG as an N-terminal His-tag fusion protein at 18°C. overnight and harvested at 30,000×g. Cells were lysed with lysozymein lysis buffer (50 mM Tris-HCl, 2 mM Tris[2-carboxyethyl] phosphine(TCEP), 10 μM ZnCl₂, 200 mM NaCl) and further treated with sonicationfor 10 min for complete lysis. The protein was purified over a Ni-NTAcolumn (Amersham) and fractions containing pure protein were pooled,concentrated and run over a sizing column (Sephadex S200 26/60 global).The peak containing soluble protein was collected and run on anIon-exchange column (MonoQ). Gradient elution (200 mM-500 mM NaCl)yielded pure protein. This protein was concentrated and dialyzed againstdialysis buffer (20 mM Tris-HCl, 2 mM TCEP) overnight. The protein wasaliquoted and frozen at −80° C. until further use.

Sirtuin modulating compounds that activated SIRT1 were identified usingthe assay described above and are shown below in Table 3. Sirtuinmodulating compounds that inhibited SIRT1 were identified using theassay described above and are shown below in Table 4. The ED₅₀ valuesfor the activating compounds are represented by A (ED₅₀=<50 μM), B(ED₅₀=51-100 μM), C (ED₅₀=101-150 μM), and D (ED₅₀=>150 μM). The ED₅₀ ofresveratrol for activation of SIRT1 is 16 μM. Similarly, the IC₅₀ valuesfor the inhibiting compounds are represented by A (IC₅₀=<50 μM), B(IC₅₀=51-100 μM), G (IC₅₀=101-150 μM), and D (C₅₀=>150 μM).

TABLE 3 COMPOUND NO STRUCTURE ED₅₀ 1

C 2

B 3

D 4

B 5

A 6

A 7

C 8

D 9

A 10

A 11

B 12

B 13

C 14

C 15

B 16

B 17

D 18

B 19

C 20

C 21

B 22

D 23

D 24

C 25

B 26

C 27

B 28

C 29

D 30

B 31

D 32

B 33

C 34

C 35

B 36

C 37

B 38

D 39

C 40

C 41

C 42

B 43

A 44

C 45

B 46

B 47

B 48

C 49

B 50

D 51

B 52

C 53

C 54

D 55

D 56

D 57

D 58

A 59

C 60

B 61

D 62

A 63

N/A 64

B 65

D 66

C 91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

TABLE 4 COMPOUND NO STRUCTURE IC₅₀ 84

ASynthesis of Compounds 91 through 110 is generally described in AngewChemie, Int. Ed. 37 (16), 2234-37, 1998 and as shown below:

Example 2 Identification of Sirtuin Modulators Using SIRT3

A fluorescence polarization assay was used to identify modulators ofSIRT3 activity. The same assay may be used to identify modulators of anysirtuin protein. The assay utilizes a peptide substrate based on afragment of Histone H4, a known sirtuin deacetylation target. Thesubstrate contains a peptide having 14 amino acid residues as follows:Biotin-GASSHSK(Ac)VLK(MR121) (SEQ ID NO: 3) wherein K(Ac) is anacetylated lysine residue. The peptide is labeled with the fluorophoreMR121 (excitation 635 nm/emission 680 nm) at the C-terminus and biotinat the N-terminus.

The peptide substrate is exposed to a sirtuin protein in the presence ofNAD⁺ to allow deacetylation of the substrate and render it sensitive tocleavage by trypsin. Trypsin is then added and the reaction is carriedto completion (i.e., the deacetylated substrate is cleaved) releasingthe MR121 fragment. Streptavidin is then added to the reaction where itcan bind both the uncleaved substrate (i.e., any remaining acetylatedsubstrate) and the non-fluorescent portion of the cleaved peptidesubstrate (i.e., the biotin containing fragment). The fluorescencepolarization signal observed for the full length peptide substrate boundto streptavidin is higher than the fluorescence polarization signalobserved for the released MR121 C-terminal fragment. Therefore, thefluorescence polarization obtained is inversely proportional to thelevel of deacetylation (e.g., the signal is inversely proportional tothe activity of the sirtuin protein). Results are read on a microplatefluorescence polarization reader (Molecular Devices Spectramax MD) withappropriate excitation and emission filters.

The fluorescence polarization assays may be conducted as follows: 0.5 μMpeptide substrate and 50 μM βNAD⁺ is incubated with 2 nM of SIRT3 for 60minutes at 37° C. in a reaction buffer (25 mM Tris-acetate pH8, 137 mMNa—Ac, 2.7 mM K—Ac, 1 mM Mg—Ac, 0.1% Pluronic F127, 10 mM CaCl₂, 1 mMTCEP, 0.025% BSA). Test compounds are solubilized in DMSO and are addedto the reaction at 11 concentrations ranging from 0.7 μM to 100 μM. TheSIRT3 protein used in the assays corresponded to amino acid residues102-399 of human SIRT3 with an N-terminal His-tag. The protein wasoverexpressed in E. coli and purified on a nickel chelate column usingstandard techniques. After the 60 minute incubation with SIRT3,nicotinamide is added to the reaction to a final concentration of 3 mMto stop the deacetylation reaction and 0.5 μg/mL of trypsin is added tocleave the deacetylated substrate. The reaction is incubated for 30minutes at 37° C. in the presence of 1 mM streptavidin. Fluorescentpolarization is determined at excitation (650 nm) and emissions (680 nm)wavelengths. The level of activity of the sirtuin protein in thepresence of the various concentrations of test compound are thendetermined and may be compared to the level of activity of the sirtuinprotein in the absence of the test compound, and/or the level ofactivity of the sirtuin proteins in the negative control (e.g., level ofinhibition) and positive control (e.g., level of activation) describedbelow.

A control for inhibition of sirtuin activity is conducted by adding 30mM nicotinamide at the start of the reaction (e.g., permitsdetermination of maximum sirtuin inhibition). A control for activationof sirtuin activity is conducted using 0.5 μg/mL of sirtuin protein toreach baseline deacetylation of the substrate (e.g., to determinenormalized sirtuin activity).

Sirtuin modulating compounds that activated SIRT3 were identified usingthe assay described above and are shown below in Table 5. Sirtuinmodulating compounds that inhibited SIRT3 were identified using theassay described above and are shown below in Table 6. The ED₅₀ valuesfor the activating compounds are represented by A (ED₅₀=<50 μM), B(ED₅₀=51-100 μM), C (ED₅₀=101-150 μM), and D (ED₅₀=>150 μM). The ED₅₀ ofresveratrol for activation of SIRT1 is 16 μM. Similarly, the IC₅₀ valuesfor the inhibiting compounds are represented by A (IC₅₀=<50 μM), B(IC₅₀=51-100 μM), C (IC₅₀=101-150 μM), and D (IC₅₀=>150 μM).

TABLE 5 COMPOUND NO STRUCTURE ED₅₀ 5

C 6

N/A 14

C 29

D 30

N/A 31

N/A 32

A 37

C 43

B 45

D 67

D 68

N/A 69

B 70

D 71

B 72

B 73

B 74

D 75

B 76

A 77

N/A 78

C 79

B 80

B 81

B 82

N/A 83

D

TABLE 6 COMPOUND NO STRUCTURE IC₅₀ 85

N/A 86

B 87

C 88

A 89

C 90

N/AEquivalents

The present invention provides among other things sirtuin-activatingcompounds and methods of use thereof. While specific embodiments of thesubject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) (www.tigr.org) and/or theNational Center for Biotechnology Information (NCBI)(www.ncbi.nlm.nih.gov).

Also incorporated by reference are the following: PCT Publications WO2005/002672; 2005/002555; and 2004/016726.

1. A compound represented by Structural Formula (Ia):

wherein: R₁₀ is selected from —CH₂-piperazinyl, —CH₂-methylpiperazinyl,—CH₂-pyrrolidyl, —CH₂-piperidyl, —CH₂-morpholino, —CH₂—N(CH₃)₂,—C(O)—NH—(CH₂)_(n)-piperazinyl, —C(O)—NH—(CH₂)_(n)-methylpiperazinyl,—C(O)—NH—(CH₂)_(n)-pyrrolidyl —C(O)—NH—(CH₂)_(n)-morpholino,—C(O)—NH—(CH₂)_(n)-piperidyl, or —C(O)—NH—(CH₂)_(n)—N(CH₃)₂, wherein nis 1 or 2; R₁₁ is selected from —C₁-C₃ straight or branched alkylene or—C(O)—; and each of ring K and ring E is independently substituted withup to three substituents independently selected from halo, —CF₃,—O—(C₁-C₃ straight or branched alkyl), —S—(C₁-C₃ straight or branchedalkyl), —N(R₄₀)(R₅₀), —S(O)₂—N(R₄₀)(R₅₀), heterocyclyl, —(C₁-C₃ straightor branched alkyl)-heterocyclyl, —O—(C₁-C₃ straight or branched alkyl)-heterocyclyl, and —S—(C₁-C₃ straight or branched alkyl)-heterocyclyl,or is optionally fused to a 5-6 membered heterocyclyl or heteroaryl,wherein any heterocyclyl or heteroaryl is optionally substituted with—C₁-C₃ straight or branched alkyl.
 2. A compound represented byStructural Formula (Ib):

wherein: Z is selected from O or S; R₁₀ is selected from —H,—C(O)—N(R₄₀)(R₅₀), —S(O)₂N(R₄₀)(R₅₀), or —CH₂—N(R₄₀)(R₅₀); wherein eachof R₄₀ and R₅₀ is independently selected from —H, —C₁-C₃ straight orbranched alkyl, —(C₁-C₃ straight or branched alkyl)—N(CH₃)₂, —(C₁-C₃straight or branched alkyl)-heterocyclyl, and —(C₁-C₃ straight orbranched alkyl)-alkylheterocyclyl, or wherein R₄₀ and R₅₀ taken togetherwith the N atom to which they are bound form a 5-6 membered heterocyclicring that is optionally substituted with —(C₁-C₃ straight or branchedalkyl), and wherein at least one of R₄₀ or R₅₀ is not H; R₁₁ is selectedfrom —C₁-C₃ straight or branched alkylene or —C(O)—; each of R₁₂ and R₁₃is independently selected from —H or —(C₁-C₃ straight or branchedalkyl), or R₁₂ and R₁₃ are taken together to form a benzene ring that issubstituted with up to two substituents independently selected from—(C₁-C₃ straight or branched alkyl), —CF₃ or halo; and ring K issubstituted with up to three substituents independently selected fromhalo, —CF₃, —O—(C₁-C₃ straight or branched alkyl), —S—(C₁-C₃ straight orbranched alkyl), —N(R₄₀)(R₅₀), —S(O)₂—N(R₄₀)(R₅₀), heterocyclyl, (C₁-C₃straight or branched alkyl)-heterocyclyl, —O—(C₁-C₃ straight or branchedalkyl)-heterocyclyl, —S—(C₁-C₃ straight or branched alkyl)-heterocyclyl,or is optionally fused to a 5-6 membered heterocyclyl or heteroaryl,wherein any heterocyclyl or heteroaryl is optionally substituted with—C₁-C₃ straight or branched alkyl.
 3. A compound represented byStructural Formula (IVa):

or a salt thereof, wherein: Ring E is optionally substituted; and R₄ andR₅ are independently —H, a substituted or unsubstituted alkyl group, asubstituted or unsubstituted aryl group or a substituted orunsubstituted non-aromatic heterocyclic group.
 4. A compound representedby Structural Formula (V):

or a salt thereof, wherein: R₄, R₅ and R₉ are independently —H, asubstituted or unsubstituted alkyl group, a substituted or unsubstitutedaryl group or a substituted or unsubstituted non-aromatic heterocyclicgroup.
 5. The compound of claim 4, wherein the compound is representedby Structural Formula (VI):

or a salt thereof, wherein: R₄, R₅ and R₉ are independently —H, asubstituted or unsubstituted alkyl group, a substituted or unsubstitutedaryl group or a substituted or unsubstituted non-aromatic heterocyclicgroup.
 6. A composition comprising a compound of any of claims 1, 2, 3,4 and 5, wherein the composition is pyrogen-free.
 7. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand a compound of any of claims 1, 2, 3, 4 and
 5. 8. A packagedpharmaceutical comprising a compound of any of claims 1, 2, 3, 4 and 5and instructions for using the compound to modulate a sirtuin.
 9. Thecompound of claim 1, wherein one of R₄₀ or R₅₀ is H.
 10. The compound ofclaim 1, wherein ring K is substituted with up to 3 substituentsindependently selected from methyl, —O-methyl, —N(CH₃)₂, or —CF₃, andwherein ring K is unsubstituted in the positions ortho to the attachmentto the rest of the molecule.
 11. The compound according to claim 1,wherein ring E is substituted with up to 2 substituents independentlyselected from methyl, —O-methyl, —S(O)₂—N(CH₃)₂, —O-methyl-morpholino,—O-ethyl-morpholino, fluoro, —CF₃, piperidyl, methylpiperidyl,pyrrolidyl, or methylpyrrolidyl.
 12. The compound according to claim 1,wherein: ring K is substituted with up to 3 substituents independentlyselected from methyl, O-methyl, N(CH₃)₂, CF₃, and wherein ring K isunsubstituted in the positions ortho to the attachment to the rest ofthe molecule; ring E is substituted with up to 2 substituentindependently selected from methyl, O-methyl, —S(O)₂—N(CH₃)₂,—O-methyl-morpholino, —O-ethyl-morpholino, fluoro, —CF₃,methylpiperidyl, or pyrrolidyl; and R₁₀ is selected from—CH₂-piperazinyl, —C(O)—NH—(CH₂)₂-piperazinyl,—C(O)—NH—(CH₂)₂-methylpiperazinyl , —C(O)—NH—(CH₂)₂-pyrrolidyl, or—C(O)—NH—(CH₂)₂—N(CH₃)₂.
 13. The compound of claim 2, wherein R₁₀ is —H.14. The compound of claim 2, wherein ring K is substituted with up to 3substituents independently selected from methyl, O-methyl, N(CH₃)₂, CF₃,and wherein ring K is unsubstituted in the positions ortho to theattachment to the rest of the molecule.
 15. The compound according toclaim 2, wherein each of R₁₂ and R₁₃ is independently selected from —H,methyl, —O-methyl, —S(O)₂—N(CH₃)₂, —O-methyl-morpholino,—O-ethyl-morpholino, fluoro, —CF₃, piperidyl, methylpiperidyl,pyrrolidyl, or methylpyrrolidyl.
 16. The compound according to claim 14,wherein each of R₁₂ and R₁₃ is methyl.